Bone mineralization proteins, DNA, vectors, expression systems

ABSTRACT

The present invention is directed to isolated nucleic acid molecules that encode LIM mineralization protein, or LMP. The invention further provides vectors comprising nucleotide sequences that encode LMP, as well as host cells comprising those vectors. Moreover, the present invention relates to methods of inducing bone formation by transfecting osteogenic precursor cells with an isolated nucleic acid molecule comprising a nucleotide sequence encoding LIM mineralization protein. The transfection may occur ex vivo or in vivo by direct injection of virus or naked plasmid DNA. In a particular embodiment, the invention provides a method of fusing a spine by transfecting osteogenic precursor cells with an isolated nucleic acid molecule having a nucleotide sequence encoding LIM mineralization protein, admixing the transfected osteogenic precursor cells with a matrix and contacting the matrix with the spine. Finally, the invention relates to methods for inducing systemic bone formation by stable transfection of host cells with the vectors of the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a division of application Ser. No. 09/721,975, filed Nov. 27,2000, which is a continuation of application Ser. No. 09/124,238, filedJul. 29, 1998, issued as U.S. Pat. No. 6,300,127 on Oct. 9, 2001, whichclaims the benefit of provisional application Nos. 60/054,219, filedJul. 30, 1997, and 60/080,407, filed Apr. 2, 1998, which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The field of the invention relates generally to osteogenic cells and theformation of bone and boney tissue in mammalian species. Specifically,the invention concerns a novel family of proteins, and nucleic acidsencoding those proteins, that enhances the efficacy of bonemineralization in vitro and in vivo. The invention provides methods fortreating a variety of pathological conditions associated bone and boneytissue, such as, for example, spine fusion, fracture repair andosteoporosis.

2. Description of the Related Art

Osteoblasts are thought to differentiate from pluripotent mesenchymalstem cells. The maturation of an osteoblast results in the secretion ofan extracellular matrix which can mineralize and form bone. Theregulation of this complex process is not well understood but is thoughtto involve a group of signaling glycoproteins known as bonemorphogenetic proteins (BMPs). These proteins have been shown to beinvolved with embryonic dorsal-ventral patterning, limb bud development,and fracture repair in adult animals. B. L. Hogan, Genes & Develop.,10:1580 (1996). This group of transforming growth factor-betasuperfamily secreted proteins has a spectrum of activities in a varietyof cell types at different stages of differentiation; differences inphysiological activity between these closely related molecules have notbeen clarified. D. M. Kingsley, Trends Genet., 10:16 (1994).

To better discern the unique physiological role of different BMPsignaling proteins, we recently compared the potency of BMP-6 with thatof BMP-2 and BMP4, for inducing rat calvarial osteoblastdifferentiation. Boden et al, Endocrinology, 137:3401 (1996). We studiedthis process in first passage (secondary) cultures of fetal rat calvariathat require BMP or glucocorticoid for initiation of differentiation. Inthis model of membranous bone formation, glucocorticoid (GC) or a BMPwill initiate differentiation to mineralized bone nodules capable ofsecreting osteocalcin, the osteoblast-specific protein. This secondaryculture system is distinct from primary rat osteoblast cultures whichundergo spontaneous differentiation. In this secondary system,glucocorticoid resulted in a ten-fold induction of BMP-6 mRNA andprotein expression which was responsible for the enhancement ofosteoblast differentiation. Boden et al., Endocrinology, 138:2920(1997).

In addition to extracellular signals, such as the BMPs, intracellularsignals or regulatory molecules may also play a role in the cascade ofevents leading to formation of new bone. One broad class ofintracellular regulatory molecules are the LIM proteins, which are sonamed because they possess a characteristic structural motif known asthe LIM domain. The LIM domain is a cysteine-rich structural motifcomposed of two special zinc fingers that are joined by a 2-amino acidspacer. Some proteins have only LIM domains, while others contain avariety of additional functional domains. LIM proteins form a diversegroup, which includes transcription factors and cytoskeletal proteins.The primary role of LIM domains appears to be in mediatingprotein-protein interactions, through the formation of dimers withidentical or different LIM domains, or by binding distinct proteins.

In LIM homeodomain proteins, that is, proteins having both LIM domainsand a homeodomain sequence, the LIM domains function as negativeregulatory elements. LIM homeodomain proteins are involved in thecontrol of cell lineage determination and the regulation ofdifferentiation, although LIM-only proteins may have similar roles.LIM-only proteins are also implicated in the control of cellproliferation since several genes encoding such proteins are associatedwith oncogenic chromosome translocations.

Humans and other mammalian species are prone to diseases or injuriesthat require the processes of bone repair and/or regeneration. Forexample, treatment of fractures would be improved by new treatmentregimens that could stimulate the natural bone repair mechanisms,thereby reducing the time required for the fractured bone to heal. Inanother example, individuals afflicted with systemic bone disorders,such as osteoporosis, would benefit from treatment regimens that wouldresults in systemic formation of new bone. Such treatment regimens wouldreduce the incidence of fractures arising from the loss of bone massthat is a characteristic of this disease.

For at least these reasons, extracellular factors, such as the BMPs,have been investigated for the purpose of using them to stimulateformation of new bone in vivo. Despite the early successes achieved withBMPs and other extracellular signalling molecules, their use entails anumber of disadvantages. For example, relatively large doses of purifiedBMPs are required to enhance the production of new bone, therebyincreasing the expense of such treatment methods. Furthermore,extracellular proteins are susceptible to degradation following theirintroduction into a host animal. In addition, because they are typicallyimmunogenic, the possibility of stimulating an immune response to theadministered proteins is ever present.

Due to such concerns, it would be desirable to have available treatmentregimens that use an intracellular signalling molecule to induce newbone formation. Advances in the field of gene therapy now make itpossible to introduce into osteogenic precursor cells, that is, cellsinvolved in bone formation, nucleotide fragments encoding intracellularsignals that form part of the bone formation process. Gene therapy forbone formation offers a number of potential advantages: (1) lowerproduction costs; (2) greater efficacy, compared to extracellulartreatment regiments, due to the ability to achieve prolonged expressionof the intracellular signal; (3) it would by-pass the possibility thattreatment with extracellular signals might be hampered due to thepresence of limiting numbers of receptors for those signals; (4) itpermits the delivery of transfected potential osteoprogenitor cellsdirectly to the site where localized bone formation is required; and (5)it would permit systemic bone formation, thereby providing a treatmentregimen for osteoporosis and other metabolic bone diseases.

SUMMARY OF THE INVENTION

The present invention seeks to overcome the drawbacks in the prior artby providing novels compositions and methods for inducing bone formationusing an intracellular signalling molecule that participates early inthe cascade of events that leads to bone formation. Applicants havediscovered 10-4/RLMP (SEQ ID NO: 1, SEQ ID NO: 2), a novel LIM gene witha sequence originally isolated from stimulated rat calvarial osteoblastcultures. The gene has been cloned, sequenced and assayed for itsability to enhance the efficacy of bone mineralization in vitro. Theprotein RLMP affects mineralization of bone matrix as well asdifferentiation of cells into the osteoblast lineage. Unlike other knowncytokines, for example, BMPs, RLMP is not a secreted protein, but isinstead an intracellular signaling molecule. This feature has theadvantage of providing intracellular signaling amplification as well aseasier assessment of transfected cells. It is also suitable for moreefficient and specific in vivo applications. Suitable clinicalapplications include enhancement of bone repair in fractures, bonedefects, bone grafting, and normal homeostasis in patients presentingwith osteoporosis.

Applicants have also cloned, sequenced and deduced the amino acidsequence of a corresponding human protein, named human LMP-1. The humanprotein demonstrates enhanced efficacy of bone mineralization in vitroand in vivo.

In addition, the applicants have characterized a truncated (short)version of LMP-1, termed HLMP-1s. This short version resulted from apoint mutation in one source of a cDNA clone, providing a stop codonwhich truncated the protein. The short version (LMP-1s) is fullyfunctional when expressed in cell culture and in vivo.

Additional features and advantages of the invention will be set forth inthe description which follows, and in part will be apparent from thedescription, or may be learned by practice of the invention. Theobjectives and other advantages of the invention will be realized andattained by the methods and compositions of matter particularly pointedout in the written description and claims hereof.

In one broad aspect, the invention relates to an isolated nucleic acidmolecule comprising a nucleic acid sequence encoding any LIMmineralization protein, wherein the nucleic acid molecule hybridizesunder standard conditions to a nucleic acid molecule complementary tothe full length of SEQ. ID NO: 25, and wherein the molecule hybridizesunder highly stringent conditions to a nucleic acid moleculecomplementary to the full length of SEQ. ID NO: 26. In a specificaspect, the isolated nucleic acid molecule encodes HLMP-1, HLMP-1s orRLMP. In addition, the invention is directed to vectors comprising thesenucleic acid molecules, as well as host cells comprising the vectors. Inanother specific aspect, the invention relates to the proteinsthemselves.

In a second broad aspect, the invention relates to antibody that isspecific for LIM mineralization protein, including HLMP-1, HLMP-1s andRLMP. In one specific ascpect, the antibody is a polyclonal antibody. Inanother specific aspect, the antibody is a monoclonal antibody.

In a third broad aspect, the invention relates to method of inducingbone formation wherein osteogenic precursor cells are transfected withan isolated nucleic acid molecule comprising a nucleotide sequenceencoding LIM mineralization protein. In one specific aspect, theisolated nucleic acid molecule is in a vector, which may be a plasmid ora virus, such as adenovirus or retrovirus. The transfection may occur exvivo or in vivo by direct injection of the isolated nucleic acidmolecule. The transfected isolated nucleic acid molecule may encodeHLMP-1, HLMP-1s or RLMP.

In a further aspect, the invention relates to methods of fusing a spineby transfecting osteogenic precursor cells with an isolated nucleic acidmolecule having a nucleotide sequence encoding LIM mineralizationprotein, admixing the transfected osteogenic precursor cells with amatrix and contacting the matrix with the spine.

In yet another aspect, the invention relates to methods for inducingsystemic bone formation by stable transfection of host cells with thevectors of the invention.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory and areintended to provide further explanation of the invention as claimed.

ABBREVIATIONS AND DEFINITIONS BMP Bone Morphogenetic Protein HLMP-1Human LMP-1, also designated as Human LIM Protein or HLMP HLMP-1s HumanLMP-1 Short (truncated) protein HLMPU Human LIM Protein Unique RegionLMP LIM mineralization protein MEM Minimal essential medium TrmTriamcinolone β-GlyP Beta-glycerolphosphate RACE Rapid Amplification ofcDNA Ends RLMP Rat LIM mineralization protein, also designated as RLMP-1RLMPU Rat LIM Protein Unique Region RNAsin RNase inhibitor ROB RatOsteoblast 10-4 Clone containing cDNA sequence for RLMP (SEQ ID NO: 2)UTR Untranslated Region

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to novel mammalian LIM proteins, hereindesignated LIM mineralization proteins, or LMP. The invention relatesmore particularly to human LMP, known as HLMP or HLMP-1. The applicantshave discovered that these proteins enhance bone mineralization inmammalian cells grown in vitro. When produced in mammals, LMP alsoinduces bone formation in, vivo.

Ex vivo transfection of bone marrow cells, osteogenic precursor cells ormesenchymal stem cells with nucleic acid that encodes LMP or HLMP,followed by reimplantation of the transfected cells in the donor, issuitable for treating a variety of bone-related disorders or injuries.For example, one can use this method to: augment long bone fracturerepair; generate bone in segmental defects; provide a bone graftsubstitute for fractures; facilitate tumor reconstruction or spinefusion; and provide a local treatment (by injection) for weak orosteoporotic bone, such as in osteoporosis of the hip, vertebrae, orwrist. Transfection with LMP or HLMP-encoding nucleic acid is alsouseful in: the percutaneous injection of transfected marrow cells toaccelerate the repair of fractured long bones; treatment of delayedunion or non-unions of long bone fractures or pseudoarthrosis of spinefusions; and for inducing new bone formation in avascular necrosis ofthe hip or knee.

In addition to ex vivo-based methods of gene therapy, transfection of arecombinant DNA vector comprising a nucleic acid sequence that encodesLMP or HLMP can be accomplished in vivo. When a DNA fragment thatencodes LMP or HLMP is inserted into an appropriate viral vector, forexample, an adenovirus vector, the viral construct can be injecteddirectly into a body site were endochondral bone formation is desired.By using a direct, percutaneous injection to introduce the LMP or HLMPsequence stimulation of bone formation can be accomplished without theneed for surgical intervention either to obtain bone marrow cells (totransfect ex vivo) or to reimplant them into the patient at the sitewhere new bone is required. Alden et al., Neurosurgical Focus (1998),have demonstrated the utility of a direct injection method of genetherapy using a cDNA that encodes BMP-2, which was cloned into anadenovirus vector.

It is also possible to carry out in vivo gene therapy by directlyinjecting into an appropriate body site, a naked, that is,unencapsulated, recombinant plasmid comprising a nucleic acid sequencethat encodes HLMP. In this embodiment of the invention, transfectionoccurs when the naked plasmid DNA is taken up, or internalized, by theappropriate target cells, which have been described. As in the case ofin vivo gene therapy using a viral construct, direct injection of nakedplasmid DNA offers the advantage that little or no surgical interventionis required. Direct gene therapy, using naked plasmid DNA that encodesthe endothelial cell mitogen VEGF (vascular endothelial growth factor),has been successfully demonstrated in human patients. Baumgartner etal., Circulation, 97(12):1114-23 (1998).

By using an adenovirus vector to deliver LMP into osteogenic cells,transient expression of LMP is achieved. This occurs because adenovirusdoes not incorporate into the genome of target cells that aretransfected. Transient expression of LMP, that is, expression thatoccurs during the lifetime of the transfected target cells, issufficient to achieve the objects of the invention. Stable expression ofLMP, however, can occur when a vector that incorporates into the genomeof the target cell is used as a delivery vehicle. Retrovirus-basedvectors, for example, are suitable for this purpose.

Stable expression of LMP is particularly useful for treating varioussystemic bone-related disorders, such as osteoporosis and osteogenesisimperfecta. For this embodiment of the invention, in addition to using avector that integrates into the genome of the target cell to deliver anLMP-encoding nucleotide sequence into target cells, LMP expression isplaced under the control of a regulatable promoter. For example, apromoter that is turned on by exposure to an exogenous inducing agent,such as tetracycline, is suitable. Using this approach, one canstimulate formation of new bone on a systemic basis by administering aneffective amount of the exogenous inducing agent. Once a sufficientquantity of bone mass is achieved, administration of the exogenousinducing agent is discontinued. This process may be repeated as neededto replace bone mass lost, for example, as a consequence ofosteoporosis.

Antibodies specific for HLMP are particularly suitable for use inmethods for assaying the osteoinductive, that is, bone-forming,potential of patient cells. In this way one can identify patients atrisk for slow or poor healing of bone repair. Also, HLMP-specificantibodies are suitable for use in marker assays to identify riskfactors in bone degenerative diseases, such as, for example,osteoporosis.

Following well known and conventional methods, the genes of the presentinvention are prepared by ligation of nucleic acid segments that encodeLMP to other nucleic acid sequences, such as cloning and/or expressionvectors. Methods needed to construct and analyze these recombinantvectors, for example, restriction endonuclease digests, cloningprotocols, mutagenesis, organic synthesis of oligonucleotides and DNAsequencing, have been described. For DNA sequencing DNA, thedieoxyterminator method is the preferred.

Many treatises on recombinant DNA methods have been published, includingSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Press, 2nd edition (1988), Davis et al., Basic Methods inMolecular Biology, Elsevier (1986), and Ausubel et al., CurrentProtocols in Molecular Biology, Wiley Interscience (1988). Thesereference manuals are specifically incorporated by reference herein.

Primer-directed amplification of DNA or cDNA is a common step in theexpression of the genes of this invention. It is typically performed bythe polymerase chain reaction (PCR). PCR is described in U.S. Pat. No.4,800,159 to Mullis et al. and other published sources. The basicprinciple of PCR is the exponential replication of a DNA sequence bysuccessive cycles of primer extension. The extension products of oneprimer, when hybridized to another primer, becomes a template for thesynthesis of another nucleic acid molecule. The primer-templatecomplexes act as substrate for DNA polymerase, which in performing itsreplication function, extends the primers. The conventional enzyme forPCR applications is the thermostable DNA polymerase isolated fromThermus aquaticus, or Taq DNA polymerase.

Numerous variations of the basic PCR method exist, and a particularprocedure of choice in any given step needed to construct therecombinant vectors of this invention is readily performed by a skilledartisan. For example, to measure cellular expression of 10-4/RLMP, RNAis extracted and reverse transcribed under standard and well knownprocedures. The resulting cDNA is then analyzed for the appropriate mRNAsequence by PCR.

The gene encoding the LIM mineralization protein is expressed in anexpression vector in a recombinant expression system. Of course, theconstructed sequence need not be the same as the original, or itscomplimentary sequence, but instead may be any sequence determined bythe degeneracy of the DNA code that nonetheless expresses an LMP havingbone forming activity. Conservative amino acid substitutions, or othermodifications, such as the occurrance of an amino-terminal methionineresidue, may also be employed.

A ribosome binding site active in the host expression system of choiceis ligated to the 5′ end of the chimeric LMP coding sequence, forming asynthetic gene. The synthetic gene can be inserted into any one of alarge variety of vectors for expression by ligating to an appropriatelylinearized plasmid. A regulatable promoter, for example, the E. coli lacpromoter, is also suitable for the expression of the chimeric codingsequences. Other suitable regulatable promoters include trp, tac, recA,T7 and lambda promoters.

DNA encoding LMP is transfected into recipient cells by one of severalstandard published procedures, for example, calcium phosphateprecipitation, DEAE-Dextran, electroporation or protoplast fusion, toform stable transformants. Calcium phosphate precipitation is preferred,particularly when performed as follows.

DNAs are coprecipitated with calcium phosphate according to the methodof Graham and Van Der, Virology, 52:456 (1973), before transfer intocells. An aliquot of 40-50 μg of DNA, with salmon sperm or calf thymusDNA as a carrier, is used for 0.5×10⁶ cells plated on a 100 mm dish. TheDNA is mixed with 0.5 ml of 2×Hepes solution (280 mM NaCl, 50 mM Hepesand 1.5 mM Na₂HPO₄, pH 7.0), to which an equal volume of 2×CaCl₂ (250 mMCaCl₂ and 10 mM Hepes, pH 7.0) is added. A white granular precipitate,appearing after 30-40 minutes, is evenly distributed dropwise on thecells, which are allowed to incubate for 4-16 hours at 37° C. The mediumis removed and the cells shocked with 15% glycerol in PBS for 3 minutes.After removing the glycerol, the cells are fed with Dulbecco's MinimalEssential Medium (DMEM) containing 10% fetal bovine serum.

DNA can also be transfected using: the DEAE-Dextran methods of Kimura etal, Virology, 49:394 (1972) and Sompayrac et al., Proc. Natl. Acad. Sci.USA, 78:7575 (1981); the electroporation method of Potter, Proc. Natl.Acad. Sci. USA, 81:7161 (1984); and the protoplast fusion method ofSandri-Goddin et al., Molec. Cell. Biol., 1:743 (1981).

Phosphoramidite chemistry in solid phase is the preferred method for theorganic synthesis of oligodeoxynucleotides and polydeoxynucleotides. Inaddition, many other organic synthesis methods are available. Thosemethods are readily adapted by those skilled in the art to theparticular sequences of the invention.

The present invention also includes nucleic acid molecules thathybridize under standard conditions to any of the nucleic acid sequencesencoding the LIM mineralization proteins of the invention. “Standardhybridization conditions” will vary iwith the size of the probe, thebackground and the concentration of the nucleic acid reagents, as wellas the type of hybridization, for example, in situ, Southern blot, orhybrization of DNA-RNA hybrids (Northern blot). The determination of“standard hybridization conditions” is within the level of skill in theart. For example, see U.S. Pat. No. 5,580,775 to Fremeau et al., hereinincorporated by reference for this purpose. See also, Southern, E. M.,J. Mol. Biol., 98:503 (1975), Alwine et al., Meth. Enzymol., 68:220(1979), and Sambrook et al., Molecular Cloning: A laboratory Manual, 2ndedition, pp. 7.19-7.50, Cold Spring Harbor Press (1989).

One preferred set of standard hybrization conditions involves a blotthat is prehybridized at 42° C. for 2 hours in 50% formamide, 5×SSPE(150 nM NaCl, 10 mM Na H₂PO₄ [pH 7.4], 1 mM EDTA [pH 8.0]), 5×Denhardt'ssolution (20 mg Ficoll, 20 mg polyvinylpyrrolidone and 20 mg BSA per 100ml water), 10% dextran sulphate, 1% SDS and 100 μg/ml salmon sperm DNA.A ³²P-labelled cDNA probe is added, and hybridization is continued for14 hours. Afterward, the blot is washed twice with 2×SSPE, 0.1% SDS for20 minutes at 22° C., followed by a 1 hour wash at 65° C. in 0.1×SSPE,0.1% SDS. The blot is then dried and exposed to x-ray film for 5 days inthe presence of an intensifying screen.

Under “highly stringent conditions,” a probe will hybridize to itstarget sequence if those two sequences are substantially identical. Asin the case of standard hybridization conditions, one of skill in theart can, given the level of skill in the art and the nature of theparticular experiment, determine the conditions under which onlysusbstantially identical sequences will hybridize.

Another aspect of the invention includes the proteins encoded by thenucleic acid sequences. In still another embodiment, the inventionrelates to the identification of such proteins based on anti-LMPantibodies. In this embodiment, protein samples are prepared for Westernblot analysis by lysing cells and separating the proteins by SDS-PAGE.The proteins are transferred to nitrocellulose by electroblotting asdescribed by Ausubel et al., Current Protocols in Molecular Biology,John Wiley and Sons (1987). After blocking the filter with instantnonfat dry milk (1 gm in 100 ml PBS), anti-LMP antibody is added to thefilter and incubated for 1 hour at room temperature. The filter iswashed thoroughly with phosphate buffered saline (PBS) and incubatedwith horseradish peroxidase (HRPO)-antibody conjugate for 1 hour at roomtemperature. The filter is again washed thoroughly with PBS and theantigen bands are identified by adding diaminobenzidine (DAB).

Monospecific antibodies are the reagent of choice in the presentinvention, and are specifically used to analyze patient cells forspecific characteristics associated with the expression of LMP.“Monospecific antibody” as used herein is defined as a single antibodyspecies or multiple antibody species with homogenous bindingcharacteristics for LMP. “Homogeneous binding” as used herein refers tothe ability of the antibody species to bind to a specific antigen orepitope, such as those associated with LMP, as described above.Monospecific antibodies to LMP are purified from mammalian antiseracontaining antibodies reactive against LMP or are prepared as monoclonalantibodies reactive with LMP using the technique of Kohler and Milstein,Nature, 256:495-97 (1975). The LMP specific antibodies are raised byimmunizing animals such as, for example, mice, rats, guinea pigs,rabbits, goats or horses, with an appropriate concentration of LMPeither with or without an immune adjuvant.

In this process, preimmune serum is collected prior to the firstimmunization. Each animal receives between about 0.1 mg and about 1000mg of LMP associated with an acceptable immune adjuvant, if desired.Such acceptable adjuvants include, but are not limited to, Freund'scomplete, Freund's incomplete, alum-precipitate, water in oil emulsioncontaining Corynebacterium parvum and tRNA adjuvants. The initialimmunization consists of LMP in, preferably, Freund's complete adjuvantinjected at multiple sites either subcutaneously (SC), intraperitoneally(IP) or both. Each animal is bled at regular intervals, preferablyweekly, to determine antibody titer. The animals may or may not receivebooster injections following the initial immunization. Those animalsreceiving booster injections are generally given an equal amount of theantigen in Freund's incomplete adjuvant by the same route. Boosterinjections are given at about three week intervals until maximal titersare obtained. At about 7 days after each booster immunization or aboutweekly after a single immunization, the animals are bled, the serumcollected, and aliquots are stored at about −20° C.

Monoclonal antibodies (mAb) reactive with LMP are prepared by immunizinginbred mice, preferably Balb/c mice, with LMP. The mice are immunized bythe IP or SC route with about 0.1 mg to about 10 mg, preferably about 1mg, of LMP in about 0.5 ml buffer or saline incorporated in an equalvolume of an acceptable adjuvant, as discussed above. Freund's completeadjuvant is preferred. The mice receive an initial immunization on day 0and are rested for about 3-30 weeks. Immunized mice are given one ormore booster immunizations of about 0.1 to about 10 mg of LMP in abuffer solution such as phosphate buffered saline by the intravenous(IV) route. Lymphocytes from antibody-positive mice, preferably spleniclymphocytes, are obtained by removing the spleens from immunized mice bystandard procedures known in the art. Hybridoma cells are produced bymixing the splenic lymphocytes with an appropriate fusion partner,preferably myeloma cells, under conditions which will allow theformation of stable hybridomas. Fusion partners may include, but are notlimited to: mouse myelomas P3/NS1/Ag 4-1; MPC-11; S-194 and Sp 2/0, withSp 2/0 being preferred. The antibody producing cells and myeloma cellsare fused in polyethylene glycol, about 1000 mol. wt., at concentrationsfrom about 30% to about 50%. Fused hybridoma cells are selected bygrowth in hypoxanthine, thymidine and aminopterin in supplementedDulbecco's Modified Eagles Medium (DMEM) by procedures known in the art.Supernatant fluids are collected from growth positive wells on aboutdays 14, 18, and 21, and are screened for antibody production by animmunoassay such as solid phase immunoradioassay (SPIRA) using LMP asthe antigen. The culture fluids are also tested in the Ouchterlonyprecipitation assay to determine the isotype of the mAb. Hybridoma cellsfrom antibody positive wells are cloned by a technique such as the softagar technique of MacPherson, “Soft Agar Techniques”, in Tissue CultureMethods and Applications, Kruse and Paterson (eds.), Academic Press(1973). See, also, Harlow et al., Antibodies: A Laboratory Manual, ColdSpring Laboratory (1988).

Monoclonal antibodies may also be produced in vivo by injection ofpristane-primed Balb/c mice, approximately 0.5 ml per mouse, with about2×10⁶ to about 6×10⁶ hybridoma cells about 4 days after priming. Ascitesfluid is collected at approximately 8-12 days after cell transfer andthe monoclonal antibodies are purified by techniques known in the art.

In vitro production in anti-LMP mAb is carried out by growing thehydridoma cell line in DMEM containing about 2% fetal calf serum toobtain sufficient quantities of the specific mAb. The mAb are purifiedby techniques known in the art.

Antibody titers of ascites or hybridoma culture fluids are determined byvarious serological or immunological assays, which include, but are notlimited to, precipitation, passive agglutination, enzyme-linkedimmunosorbent antibody (ELISA) technique and radioimmunoassay (RIA)techniques. Similar assays are used to detect the presence of the LMP inbody fluids or tissue and cell extracts.

It is readily apparent to those skilled in the art that the abovedescribed methods for producing monospecific antibodies may be utilizedto produce antibodies specific for polypeptide fragments of LMP,full-length nascent LMP polypeptide, or variants or alleles thereof.

On Jul. 22, 1997, a sample of 10-4/RLMP in a vector designatedpCMV2/RLMP (which is vector pRc/CMV2 with insert 10-4 clone/RLMP) wasdeposited with the American Type Culture Collection (ATCC), 12301Parklawn Drive, Rockville, Md. 20852. The culture accession number forthat deposit is 209153. On Mar. 19, 1998, a sample of the vector pHis-Awith insert HLPM-1s was deposited at the American Type CultureCollection. The culture accession number for that deposit is 209698.These deposits, made under the requirements of the Budapest Treaty, willbe maintained in the ATCC for at least 30 years and will be madeavailable to the public upon the grant of a patent disclosing them. Itshould be understood that the availability of a deposit does notconstitute a license to practice the subject invention in derogation ofpatent rights granted by government action.

In assessing the nucleic acids, proteins, or antibodies of theinvention, enzyme assays, protein purification, and other conventionalbiochemical methods are employed. DNA and RNA are analyzed by Southernblotting and Northern blotting techniques, respectively. Typically, thesamples analyzed are size fractionated by gel electrophoresis. The DNAor RNA in the gels are then transferred to nitrocellulose or nylonmembranes. The blots, which are replicas of sample patterns in the gels,were then hybridized with probes. Typically, the probes areradiolabelled, preferably with ³²P, although one could label the probeswith other signal-generating molecules known to those in the art.Specific bands of interest can then be visualized by detection systems,such as autoradiography.

For purposes of illustrating preferred embodiments of the presentinvention, the following, non-limiting examples are included. Theseresults demonstrate the feasibiliity of inducing or enhancing theformation of bone using the LIM mineralization proteins of theinvention, and the isolated nucleic acid molecules encoding thoseproteins.

EXAMPLE 1 Calvarial Cell Culture

Rat calvarial cells, also known as rat osteoblasts (“ROB”), wereobtained from 20-day pre-parturition rats as previously described. Bodenet al., Endocrinology, 137(8):3401-07 (1996). Primary cultures weregrown to confluence (7 days), trypsinized, and passed into 6-well plates(1×10⁵ cells/35 mm well) as first subculture cells. The subculturecells, which were confluent at day 0, were grown for an additional 7days. Beginning on day 0, media were changed and treatments (Trm and/orBMPs) were applied, under a laminar flow hood, every 3 or 4 days. Thestandard culture protocol was as follows: days 1-7, MEM, 10% FBS, 50μg/ml ascorbic acid, ±stimulus; days 8-14, BGJb medium, 10% FBS, 5 mMβ-GlyP (as a source of inorganic phosphate to permit mineralization).Endpoint analysis of bone nodule formation and osteocalcin secretion wasperformed at day 14. The dose of BMP was chosen as 50 ng/ml based onpilot experiments in this system that demonstrated a mid-range effect onthe dose-response curve for all BMPs studied.

EXAMPLE 2 Antisense Treatment and Cell Culture

To explore the potential functional role of LMP-1 during membranous boneformation, we synthesized an antisense oligonucleotide to block LMP-1mRNA translation and treated secondary osteoblast cultures that wereundergoing differentiation initiated by glucocorticoid. Inhibition ofRLMP expression was accomplished with a highly specific antisenseoligonucleotide (having no significant homologies to known ratsequences) corresponding to a 25 bp sequence spanning the putativetranslational start site (SEQ ID NO: 35). Control cultures either didnot receive oligonucleotide or they received sense oligonucleotide.Experiments were performed in the presence (preincubation) and absenceof lipofectamine. Briefly, 22 μg of sense or antisense RLMPoligonucleotide was incubated in MEM for 45 minutes at room temperature.Following that incubation, either more MEM or pre-incubatedlipofectamine/MEM (7% v/v; incubated 45 minutes at room temperature) wasadded to achieve an oligonucleotide concentration of 0.2 μM. Theresulting mixture was incubated for 15 minutes at room temperature.Oligonucleotide mixtures were then mixed with the appropriate medium,that is, MEM/Ascorbate/±Trm, to achieve a final oligonucleotideconcentration of 0.1 μM.

Cells were incubated with the appropriate medium (±stimulus) in thepresence or absence of the appropriate oligonucleotides. Culturesoriginally incubated with lipofectamine were re-fed after 4 hours ofincubation (37° C.; 5% CO₂) with media containing neither lipofectaminenor oligonucleotide. All cultures, especially cultures receivingoligonucleotide, were re-fed every 24 hours to maintain oligonucleotidelevels.

LMP-1 antisense oligonucleotide inhibited mineralized nodule formationand osteocalcin secretion in a dose-dependent manner, similar to theeffect of BMP-6 oligonucleotide. The LMP-1 antisense block in osteoblastdifferentiation could not be rescued by addition of exogenous BMP-6,while the BMP-6 antisense oligonucleotide inhibition was reversed withaddition of BMP-6. This experiment further confirmed the upstreamposition of LMP-1 relative to BMP-6 in the osteoblast differentiationpathway. LMP-1 antisense oligonucleotide also inhibited spontaneousosteoblast differentiation in primary rat osteoblast cultures.

EXAMPLE 3 Quantitation of Mineralized Bone Nodule Formation

Cultures of ROBs prepared according to Examples 1 and 2 were fixedovernight in 70% ethanol and stained with von Kossa silver stain. Asemi-automated computerized video image analysis system was used toquantitate nodule count and nodule area in each well. Boden et al.,Endocrinology, 137(8):3401-07 (1996). These values were then divided tocalculate the area per nodule values. This automated process wasvalidated against a manual counting technique and demonstrated acorrelation coefficient of 0.92 (p<0.000001). All data are expressed asthe mean +standard error of the mean (S.E.M.) calculated from 5 or 6wells at each condition. Each experiment was confirmed at least twiceusing cells from different calvarial preparations.

EXAMPLE 4 Quantitation of Osteocalcin Secretion

Osteocalcin levels in the culture media were measured using acompetitive radioimmunoassay with a monospecific polyclonal antibody(Pab) raised in our laboratory against the C-terminal nonapeptide of ratosteocalcin as described in Nanes et al., Endocrinology, 127:588 (1990).Briefly, 1 μg of nonapeptide was iodinated with 1 mCi ¹²⁵I-Na by thelactoperoxidase method. Tubes containing 200 μl of assay buffer (0.02Msodium phosphate, 1 mM EDTA, 0.001% thimerosal, 0.025% BSA) receivedmedia taken from cell cultures or osteocalcin standards (0-12,000 fmole)at 100 μl/tube in assay buffer. The Pab (1:40,000; 100 μl) was thenadded, followed by the iodinated peptide (12,000 cpm; 100 μl). Samplestested for non-specific binding were prepared similarly but contained noantibody.

Bound and free PAbs were separated by the addition of 700 μl goatanti-rabbit IgG, followed by incubation for 18 hours at 4° C. Aftersamples were centrifuged at 1200 rpm for 45 minutes, the supernatantswere decanted and the precipitates counted in a gamma counter.Osteocalcin values were reported in fmole/100 μl, which was thenconverted to pmole/ml medium (3-day production) by dividing those valuesby 100. Values were expressed as the mean±S.E.M. of triplicatedeterminations for 5-6 wells for each condition. Each experiment wasconfirmed at least two times using cells from different calvarialpreparations.

EXAMPLE 5 Effect of Trm and RLMP on Mineralization In Vitro

There was little apparent effect of either the sense or antisenseoligonucleotides on the overall production of bone nodules in thenon-stimulated cell culture system. When ROBs were stimulated with Trm,however, the antisense oligonucleotide to RLMP inhibited mineralizationof nodules by >95%. The addition of exogenous BMP-6 to theoligonucleotide-treated cultures did not rescue the mineralization ofRLMP-antisense-treated nodules.

Osteocalcin has long been synonymous with bone mineralization, andosteocalcin levels have been correlated with nodule production andmineralization. The RLMP-antisense oligonucleotide significantlydecreases osteocalcin production, but the nodule count inantisense-treated cultures does not change significantly. In this case,the addition of exogenous BMP-6 only rescued the production ofosteocalcin in RLMP-antisense-treated cultures by 10-15%. This suggeststhat the action of RLMP is downstream of, and more specific than, BMP-6.

EXAMPLE 6 Harvest and Purification of RNA

Cellular RNA from duplicate wells of ROBs (prepared according toExamples 1 and 2 in 6-well culture dishes) was harvested using 4Mguanidine isothiocyanate (GIT) solution to yield statisticaltriplicates. Briefly, culture supernatant was aspirated from the wells,which were then overlayed with 0.6 ml of GIT solution per duplicate wellharvest. After adding the GIT solution, the plates were swirled for 5-10seconds (being as consistent as possible). Samples were saved at −70° C.for up to 7 days before further processing.

RNA was purified by a slight modification of standard methods accordingto Sambrook et al, Molecular Cloning: a Laboratory Manual, 2nd Ed.,chapter 7.19, Cold Spring Harbor Press (1989). Briefly, thawed samplesreceived 60 μl 2.0M sodium acetate (pH 4.0), 550 μl phenol (watersaturated) and 150 μl chloroform:isoamyl alcohol (49:1). Aftervortexing, the samples were centrifuged (10000×g; 20 minutes; 4° C.),the aqueous phase transferred to a fresh tube, 600 μl isopropanol wasadded and the RNA precipitated overnight at −20° C.

Following the overnight incubation, the samples were centrifuged(10000×g; 20 minutes) and the supernatant was aspirated gently. Thepellets were resuspended in 400 μl DEPC-treated water, extracted oncewith phenol:chloroform (1:1), extracted with chloroform:isoamyl alcohol(24:1) and precipitated overnight at −20° C. after addition of 40 μlsodium acetate (3.0M; pH 5.2) and 1.0 ml absolute ethanol. To recoverthe cellular RNA, the samples were centrifuged (10000×g; 20 min), washedonce with 70% ethanol, air dried for 5-10 minutes and resuspended in 20μl of DEPC-treated water. RNA concentrations were calculated fromoptical densities that were determined with a spectrophotometer.

EXAMPLE 7 Reverse Transcription-Polymerase Chain Reaction

Heated total RNA (5 μg in 10.5 μl total volume DEPC-H₂O at 65° C. for 5minutes) was added to tubes containing 4 μl 5×MMLV-RT buffer, 2 μldNTPs, 2 μl dT17 primer (10 pmol/ml), 0.5 μl RNAsin (40 U/ml) and 1 μlMMLV-RT (200 units/μl). The samples were incubated at 37° C. for 1 hour,then at 95° C. for 5 minutes to inactivate the MMLV-RT. The samples werediluted by addition of 80 μl of water.

Reverse-transcribed samples (5 μl) were subjected to polymerase-chainreaction using standard methodologies (50 μl total volume). Briefly,samples were added to tubes containing water and appropriate amounts ofPCR buffer, 25 mM MgCl₂, dNTPs, forward and reverse primers forglyceraldehyde 3-phosphate dehydrogenase (GAP, a housekeeping gene)and/or BMP-6), ³²P-dCTP, and Taq polymerase. Unless otherwise noted,primers were standardized to run consistently at 22 cycles (94° C., 30″;58° C., 30″; 72° C., 20″).

EXAMPLE 8 Quantitation of RT-PCR Products by Polyacrylamide GelElectrophoresis (PAGE) and Phosphorlmager Analysis

RT-PCR products received 5 μl/tube loading dye, were mixed, heated at65° C. for 10 min and centrifuged. Ten μl of each reaction was subjectedto PAGE (12% polyacrylamide:bis; 15 V/well; constant current) understandard conditions. Gels were then incubated in gel preserving buffer(10% v/v glycerol, 7% v/v acetic acid, 40% v/v methanol, 43% deionizedwater) for 30 minutes, dried (80° C.) in vacuo for 1-2 hours anddeveloped with an electronically-enhanced phosphoresence imaging systemfor 6-24 hours. Visualized bands were analyzed. Counts per band wereplotted graphically.

EXAMPLE 9 Differential Display PCR

RNA was extracted from cells stimulated with glucocorticoid (Trm, 1 nM).Heated, DNase-treated total RNA (5 μg in 10.5 μl total volume inDEPC-H₂O at 65° C. for 5 minutes) was reverse transcribed as describedin Example 7, but H-T₁₁M (SEQ ID. NO: 4) was used as the MMLV-RT primer.The resulting cDNAs were PCR-amplified as described above, but withvarious commercial primer sets (for example, H-T₁₁G (SEQ ID NO: 4) andH-AP-10 (SEQ ID. NO: 5); GenHunter Corp, Nashville, Tenn.).Radiolabelled PCR products were fractionated by gel electrophoresis on aDNA sequencing gel. After electrophoresis, the resulting gels were driedin vacuo and autoradiographs were exposed overnight. Bands representingdifferentially-expressed cDNAs were excised from the gel and reamplifiedby PCR using the method of Conner et al., Proc. Natl. Acad. Sci. USA,88:278 (1983). The products of PCR reamplification were cloned into thevector PCR-II (TA cloning kit; InVitrogen, Carlsbad, Calif.).

EXAMPLE 10 Screening of a UMR 106 Rat Osteosarcoma Cell cDNA Library

A UMR 106 library (2.5×10¹⁰ pfu/ml) was plated at 5×10⁴ pfu/ml onto agarplates (LB bottom agar) and the plates were incubated overnight at 370°C. Filter membranes were overlaid onto plates for two minutes. Onceremoved, the filters were denatured, rinsed, dried and UV cross-linked.The filters were then incubated in pre-hyridization buffer (2× PIPES [pH6.5], 5% formamide, 1% SDS and 100 μg/ml denatured salmon sperm DNA) for2 h at 42° C. A 260 base-pair radiolabelled probe (SEQ ID NO: 3; ³²Plabelled by random priming) was added to the entire hybridizationmix/filters, followed by hybridization for 18 hours at 42° C. Themembranes were washed once at room temperature (10 min, 1×SSC, 0.1% SDS)and three times at 55° C. (15 min, 0.1×SSC, 0.1% SDS).

After they were washed, the membranes were analyzed by autoradiographyas described above. Positive clones were plaque purified. The procedurewas repeated with a second filter for four minutes to minimize spuriouspositives. Plaque-purified clones were rescued as lambda SK(−)phagemids. Cloned cDNAs were sequenced as described below.

EXAMPLE 11 Sequencing of Clones

Cloned cDNA inserts were sequenced by standard methods. Ausubel et al,Current Protocols in Molecular Biology, Wiley lnterscience (1988).Briefly, appropriate concentrations of termination mixture, template andreaction mixture were subjected to !an appropriate cycling protocol (95°C.,30s; 680° C.,30s; 72° C.,60s;×25). Stop mixture was added toterminate the sequencing reactions. After heating at 920° C. for 3minutes, the samples were loaded onto a denaturing 6% polyacrylamidesequencing gel (29:1 volts, constant current. After electrophoresis, thegels were dried in vacuo and autoradiographed.

The autoradiographs were analyzed manually. The resulting sequences werescreened against the databases maintained by the National Center forBiotechnology Information (NIH, Bethesda, Md.) using the BLASTn programset with default parameters. Based on the sequence data, new sequencingprimers were prepared and the process was repeated until the entire genehad been sequenced. All sequences were confirmed a minimum of threetimes in both orientations.

Nucleotide and amino acid sequences were also analyzed using the PCGENEsoftware package (version 16.0). Per cent homology values for nucleotidesequences were calculated by the program NALIGN, using the followingparameters: weight of non-matching nucleotides, 10; weight ofnon-matching gaps, 10; maximum number of nucleotides considered, 50; andminimum number of nucleotides considered, 50.

For amino acid sequences, per cent homology values were calculated usingPALIGN. A value of 10 was selected for both the open gap cost and theunit gap cost.

EXAMPLE 12 Cloning of RLMP cDNA

The differential display PCR amplification products described in Example9 contained a major band of approximately 260 base pairs. This sequencewas used to screen a rat osteosarcoma (UMR 106) cDNA library. Positiveclones were subjected to nested primer analysis to obtain the primersequences necessary for amplifying the full length cDNA. (SEQ. ID NOs:11, 12, 29, 30 and 31) One of those positive clones selected for furtherstudy was designated clone 10-4.

Sequence analysis of the full-length cDNA in clone 10-4, determined bynested primer analysis, showed that clone 10-4 contained the original260 base-pair fragment identified by differential display PCR. Clone10-4 (1696 base pairs; SEQ ID NO: 2) contains an open reading frame of1371 base pairs encoding a protein having 457 amino acids (SEQ ID NO:1). The termination codon, TGA, occurs at nucleotides 1444-1446. Thepolyadenylation signal at nucleotides 1675-1680, and adjacent poly(A)⁺tail, was present in the 3′ noncoding region. There were two potentialN-glycosylation sites, Asn-Lys-Thr and Asn-Arg-Thr, at amino acidpositions 113-116 and 257-259 in SEQ ID NO: 1, respectively. Twopotential cAMP- and cGMP-dependent protein kinase phosphorylation sites,Ser and Thr, were found at amino acid positions 191 and 349,respectively. There were five potential protein kinase C phosphorylationsites, Ser or Thr, at amino acid positions 3, 115, 166, 219, 442. Onepotential ATP/GTP binding site motif A (P-loop),Gly-Gly-Ser-Asn-Asn-Gly-Lys-Thr, was determined at amino acid positions272-279.

In addition, two highly conserved putative LIM domains were found atamino acid positions 341-391 and 400-451. The putative LIM domains inthis newly identified rat cDNA clone showed considerable homology withthe LIM domains of other known LIM proteins. However, the overallhomology with other rat LIM proteins was less than 25%. RLMP (alsodesignated 10-4) has 78.5% amino acid homology to the human enigmaprotein (see U.S. Pat. No. 5,504,192), but only 24.5% and 22.7% aminoacid homology to its closest rat homologs, CLP-36 and RIT-18,respectively.

EXAMPLE 13 Northern Blot Analysis of RLMP Expression

Thirty μg of total RNA from ROBs, prepared according to Examples 1 and2, was size fractionated by formaldehyde gel electrophoresis in 1%agarose flatbed gels and osmotically transblotted to nylon membranes.The blot was probed with a 600 base pair EcoR1 fragment of full-length10-4 cDNA labeled with ³²P-dCTP by random priming.

Northern blot analysis showed a 1.7 kb mRNA species that hybridized withthe RLMP probe. RLMP mRNA was up-regulated approximately 3.7-fold inROBs after 24 hours exposure to BMP-6. No up-regulation of RMLPexpression was seen in BMP-2 or BMP4-stimulated ROBs at 24 hours.

EXAMPLE 14 Statistical Methods

For each reported nodulelosteocalcin result, data from 5-6 wells from arepresentative experiment were used to calculate the mean±S.E.M. Graphsmay be shown with data normalized to the maximum value for eachparameter to allow simultaneous graphing of nodule counts, mineralizedareas and osteocalcin.

For each reported RT-PCR, RNase protection assay or Western blotanalysis, data from triplicate samples of representative experiments,were used to determine the mean±S.E.M. Graphs may be shown normalized toeither day 0 or negative controls and expressed as fold-increase abovecontrol values.

Statistical significance was evaluated using a one-way analysis ofvariance with post-hoc multiple comparison corrections of Bonferroni asappropriate. D. V. Huntsberger, “The Analysis of Variance,” in Elementsof Statistical Variance, P. Billingsley (ed.), pp. 298-330, Allyn &Bacon Inc., Boston, Mass. (1977) and Sigmastat, Jandel Scientific, CorteMadera, Calif. Alpha levels for significance were defined as p<0.05.

EXAMPLE 15 Detection of Rat LIM Mineralization Protein by Western BlotAnalysis

Polyclonal antibodies were prepared according to the methods of Englandet al., Biochim. Biophys. Acta, 623:171 (1980) and Timmer et al., J.Biol. Chem., 268:24863 (1993).

HeLa cells were transfected with pCMV2/RLMP. Protein was harvested fromthe transfected cells according to the method of Hair et al., LeukemiaResearch, 20:1 (1996). Western Blot Analysis of native RLMP wasperformed as described by Towbin et al., Proc. Natl. Acad. Sci. USA,76:4350 (1979).

EXAMPLE 16 Synthesis of the Rat LMP-Unique (RLMPU) Derived Human PCRProduct

Based on the sequence of the rat LMP-1 cDNA, forward and reverse PCRprimers (SEQ ID NOs: 15 and 16) were synthesized and a unique 223base-pair sequence was PCR amplified from the rat LMP-1 cDNA. A similarPCR product was isolated from human MG63 osteosarcoma cell cDNA with thesame PCR primers. RNA was harvested from MG63 osteosarcoma cells grownin T-75 flasks. Culture supernatant was removed by aspiration and theflasks were overlayed with 3.0 ml of GIT solution per duplicate, swirledfor 5-10 seconds, and the resulting solution was transferred to 1.5 mleppendorf tubes (5 tubes with 0.6 ml/tube). RNA was purified by a slightmodification of standard methods, for example, see Sambrook et al.,Molecular Cloning: A Laboratory Manual, chapter 7, page 19, Cold SpringHarbor Laboratory Press (1989) and Boden et al., Endocrinology,138:2820-28 (1997). Briefly, the 0.6 ml samples received 60 μl 2.0Msodium acetate (pH 4.0), 550 μl water saturated phenol and 150 μlchloroform:isoamyl alcohol (49:1). After addiiton of those reagents, thesamples were vortexed, centrifuged (10000×g; 20 min; 4° C.) and theaqueous phase transferred to a fresh tube. Isopropanol (600 μl) wasadded and the RNA was precipitated overnight at −20° C. The samples werecentrifuged (10000×g; 20 minutes) and the supernatant was aspiratedgently. The pellets were resuspended in 400 μl of DEPC-treated water,extracted once with phenol:chloroform (1:1), extracted withchloroform;isoamyl alcohol (24:1) and precipitated overnight at −20° C.in 40 μl sodium acetate (3.0M; pH 5.2) and 1.0 ml absolute ethanol.After precipitation, the samples were centrifuged (10000×g; 20 min),washed once with 70% ethanol, air dried for 5-10 minutes and resuspendedin 20 μl of DEPC-treated water. RNA concentrations were derived fromoptical densities.

Total RNA (5 μl in 10.5 μL total volume in DEPC-H₂O) was heated at 65°C. for 5 minutes, and then added to tubes containing 4 μl 5× MMLV-RTbuffer, 2 μl dNTPs, 2 μl dT17 primer (10 pmol/ml), 0.5 μl RNAsin (40U/ml) and 1 μl MMLV-RT (200 units/μl). The reactions were incubated at37° C. for 1 hour. Afterward, the MMLV-RT was inactivated by heating at95° C. for 5 minutes. The samples were diluted by addition of 80 μlwater.

Transcribed samples (5 μl) were subjected to polymerase-chain reactionusing standard methodologies (50 μl total volume). Boden et al.,Endocrinology, 138:2820-28 (1997); Ausubel et al., “Quantitation of rareDNAs by the polymerase chain reaction”, in Current Protocols inMolecular Biology, chapter 15.31-1, Wiley & Sons, Trenton, N.J. (1990).Briefly, samples were added to tubes containing water and appropriateamounts of PCR buffer (25 mM MgCl₂, dNTPs, forward and reverse primers(for RLMPU; SEQ ID NOs: 15 and 16), ³²P-dCTP, and DNA polymerase.Primers were designed to run consistently at 22 cycles for radioactiveband detection and 33 cycles for amplification of PCR product for use asa screening probe (94° C., 30 sec, 58° C., 30 sec; 72° C., 20 sec).

Sequencing of the agarose gel-purified MG63 osteosarcoma-derived PCRproduct gave a sequence more than 95% homologous to the RLMPU PCRproduct. That sequence is designated HLMP unique region (HLMPU; SEQ IDNO: 6).

EXAMPLE 17 Screening of Reverse-transcriptase-derived MG63 cDNA

Screening was performed with PCR using specific primers (SEQ ID NOs: 16and 17) as described in Example 7. A 717 base-pair MG63 PCR product wasagarose gel purified and sequenced with the given primers (SEQ. ID NOs:12, 15, 16, 17, 18, 27 and 28). Sequences were confirmed a minimum oftwo times in both directions. The MG63 sequences were aligned againsteach other and then against the full-length rat LMP cDNA sequence toobtain a partial human LMP cDNA sequence (SEQ ID NO: 7).

EXAMPLE 18 Screening of a Human Heart cDNA Library

Based on Northern blot experiments, it was determined that LMP-1 isexpressed at different levels by several different tissues, includinghuman heart muscle. A human heart cDNA library was therefore examined.The library was plated at 5×10⁴ pfu/ml onto agar plates (LB bottom agar)and plates were grown overnight at 37° C. Filter membranes were overlaidonto the plates for two minutes. Afterward, the filters denatured,rinsed, dried, UV cross-linked and incubated in pre-hyridization buffer(2×PIPES [pH 6.5]; 5% formamide, 1% SDS, 100 g/ml denatured salmon spermDNA) for 2 h at 42° C. A radiolabelled, LMP-unique, 223 base-pair probe(³²P, random primer labelling; SEQ ID NO: 6) was added and hybridizedfor 18 h at 42° C. Following hybridization, the membranes were washedonce at room temperature (10 min, 1×SSC, 0.1% SDS) and three times at55° C. (15 min, 0.1×SSC, 0.1% SDS). Double-positive plaque-purifiedheart library clones, identified by autoradiography, were rescued aslambda phagemids according to the manufacturers' protocols (Stratagene,La Jolla, Calif.).

Restriction digests of positive clones yielded cDNA inserts of varyingsizes. Inserts greater than 600 base-pairs in length were selected forinitial screening by sequencing. Those inserts were sequenced bystandard methods as described in Example 11.

One clone, number 7, was also subjected to automated sequence analysisusing primers corresponding to SEQ ID NOs: 11-14, 16 and 27. Thesequences obtained by these methods were routinely 97-100% homologous.Clone 7 (Partial Human LMP-1 cDNA from a heart library; SEQ. ID NO: 8)contained sequence that was more than 87% homologous to the rat LMP cDNAsequence in the translated region.

EXAMPLE 19 Determination of Full-Length Human LMP-1 cDNA

Overlapping regions of the MG63 human osteosarcoma cell CDNA sequenceand the human heart cDNA clone 7 sequence were used to align those twosequences and .derive a complete human cDNA sequence of 1644 basepairs.NALIGN, a program in the PCGENE software package, was used to align thetwo sequences. The overlapping regions of the two sequences constitutedapproximately 360 base-pairs having complete homology except for asingle nucleotide substitution at nucleotide 672 in the MG63 cDNA (SEQID NO: 7) with clone 7 having an “A” instead of a “G” at thecorresponding nucleotide 516 (SEQ ID NO: 8).

The two aligned sequences were joined using SEQIN, another subprogram ofPCGENE, using the “G” substitution of the MG63 osteosarcoma cDNA clone.The resulting sequence is shown in SEQ ID NO: 9. Alignment of the novelhuman-derived resulting sequence with the rat LMP-1 cDNA wasaccomplished with NALIGN. The full-length human LMP-1 cDNA sequence(SEQ. ID NO: 9) is 87.3% homologous to the translated portion of ratLMP-1 cDNA sequence.

EXAMPLE 20 Determination of Amino Acid Sequence of Human LMP-1

The putative amino acid sequence of human LMP-1 was determined with thePCGENE subprogram TRANSL. The open reading frame in SEQ ID NO: 9 encodesa protein comprising 457 amino acids (SEQ. ID NO: 10). Using the PCGENEsubprogram Palign, the human LMP-1 amino acid sequence was found to be94.1% homologous to the rat LMP-1 amino acid sequence.

EXAMPLE 21 Determination of the 5 Prime Untranslated Region of the HumanLMP cDNA

MG63 5′ cDNA was amplified by nested RT-PCR of MG63 total RNA using a 5′rapid amplification of cDNA ends (5′ RACE) protocol. This methodincluded first strand cDNA synthesis using a lock-docking oligo (dT)primer with two degenerate nucleotide positions at the 3′ end (Chenchiket al., CLONTECHniques. X:5 (1995); Borson et al., PC Methods Applic.,2:144 (1993)). Second-strand synthesis is performed according to themethod of Gubler et al., Gene. 25:263 (1983), with a cocktail ofEscherichia coli DNA polymerase I, RNase H, and E. coli DNA ligase.After creation of blunt ends with T4 DNA polymerase, double-strandedcDNA was ligated to the fragment(5′-CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT-3′) (SEQ.ID NO: 19).Prior to RACE, the adaptor-ligated cDNA was diluted to a concentrationsuitable for Marathon RACE reactions (1:50). Adaptor-ligateddouble-stranded cDNA was then ready to be specifically cloned.

First-round PCR was performed with the adaptor-specific oligonucleotide,5′′-CCATCCTAATACGACTCACTATAGGGC-3′ (AP1) (SEQ.ID NO: 20) as sense primerand a Gene Specific Primer (GSP) from the unique region described inExample 16 (HLMPU). The second round of PCR was performed using a nestedprimers GSP1-HLMPU (antisense/reverse primer) (SEQ. ID NO: 23) andGSP2-HLMPUF (SEQ. ID NO: 24) (see Example 16; sense/forward primer). PCRwas performed using a commercial kit (Advantage cDNA PCR core kit;CloneTech Laboratories Inc., Palo Alto, Calif.) that utilizes anantibody-mediated, but otherwise standard, hot-start protocol. PCRconditions for MG63 cDNA included an initial hot-start denaturation (94°C., 60 sec) followed by: 94° C., 30 sec; 60° C., 30 sec; 68° C., 4 min;30 cycles. The first-round PC product was approximately 750 base-pairsin length whereas the nested PCR product was approximately 230base-pairs. The first-round PCR product was cloned into linearized pCR2.1 vector (3.9 Kb). The inserts were sequenced in both directions usingM13 Forward and Reverse primers (SEQ. ID NO: 11; SEQ. ID NO: 12)

EXAMPLE 22 Determination of Full-length Human LMP-1 cDNA with 5 PrimeUTR

Overlapping MG63 human osteosarcoma cell cDNA 5′-UTR sequence (SEQ IDNO: 21), MG63 717 base-pair sequence (Example 17; SEQ ID NO: 8) andhuman heart cDNA clone 7 sequence (Example 18) were aligned to derive anovel human cDNA sequence of 1704 base-pairs (SEQ.ID NO: 22). Thealignment was accomplished with NALIGN, (both PCGENE and Omiga 1.0;Intelligenetics). Over-lapping sequences constituted nearly the entire717 base-pair region (Example 17) with 100% homology.

Joining of the aligned sequences was accomplished with SEQIN.

EXAMPLE 23 Construction of LIM Protein Expression Vector

The construction of pHIS-5ATG LMP-1s expression vector was carried outwith the sequences described in Examples 17 and 18. The 717 base-pairclone (Example 17; SEQ ID NO: 7) was digested with ClaI and EcoRV. Asmall fragment (˜250 base-pairs) was gel purified. Clone 7 (Example 18;SEQ ID NO: 8) was digested with ClaI and XbaI and a 1400 base-pairfragment was gel purified. The isolated 250 base-pair and 1400 base-pairrestriction fragments were ligated to form a fragment of ˜1650base-pairs.

Due to the single nucleotide substitution in Clone 7 (relative to the717 base-pair PCR sequence and the original rat sequence) a stop codonat translated base-pair 672 resulted. Because of this stop codon, atruncated (short) protein was encoded, hence the name LMP-1s. This wasthe construct used in the expression vector (SEQ ID NO: 32). The fulllength cDNA sequence with 5′ UTR (SEQ ID NO: 33) was created byalignment of SEQ ID NO: 32 with the 5′ RACE sequence (SEQ ID NO: 21).The amino protein and confirmed by Western blot (as in Example 15) torun at the predicted molecular weight of ˜23.7 kD.

The pHis-ATG vector (InVitrogen, Carlsbad, Calif.) was digested withEcoRV and XbaI. The vector was recovered and the 650 base-pairrestriction fragment was then ligated into the linearized pHis-ATG. Theligated product was cloned and amplified.

The pHis-ATG-LMP-1s Expression vector, also designated pHIS-A withinsert HLMP-1s, was purified by standard methods.

EXAMPLE 24 Induction of Bone Nodule Formation and Mineralization InVitro with LMP Expression Vector

Rat Calvarial cells were isolated and grown in secondary cultureaccording to Example 1. Cultures were either unstimulated or stimulatedwith glucocorticoid (GC) as described in Example 1. A modification ofthe Superfect Reagent (Qiagen, Valencia, Calif.) transfection protocolwas used to transfect 3 μg/well of each vector into secondary ratcalvarial osteoblast cultures according to Example 25. Mineralizednodules were visualized by Von Kossa staining, as described in Example3.

Human LMP-1s gene product overexpression alone induced bone noduleformation (˜203 nodules/well) in vitro. Levels of nodules wereapproximately 50% of those induced by the GC positive control (˜412nodules/well). Other positive controls included the pHisA-LMP-Ratexpression vector (˜152 nodules/well) and the pCMV2/LMP-Rat-FwdExpression vector (˜206 nodules/well), whereas the negative controlsincluded the pCMV2/LMP-Rat-Rev. Expression vector (˜2 nodules/well) anduntreated (NT) plates (˜4 nodules/well). These data demonstrate that thehuman cDNA was at least as osteoinductive as the rat cDNA. The effectwas less than that observed with GC stimulation, most likely due tosuboptimal doses of Expression vector.

EXAMPLE 25 LMP-Induced Cell Differentiation In Vitro and In Vivo

The rat LMP cDNA in clone 10-4 (see Example 12) was excised from thevector by double-digesting the clone with NotI and ApaI overnight at 37°C. Vector pCMV2 MCS (InVitrogen, Carlsbad, Calif.) was digested with thesame restriction enzymes. Both the linear cDNA fragment from clone 10-4and pCMV2 were gel purified, extracted and ligated with T4 ligase. Theligated DNA was gel purified, extracted and used to transform E. coliJM109 cells for amplification. Positive agar colonies were picked,digested with NotI and ApaI and the restriction digests were examined bygel electrophoresis. Stock cultures were prepared of positive clones.

A reverse vector was prepared in analogous fashion except that therestriction enzymes used were XbaI and HindIII. Because theserestriction enzymes were used, the LMP cDNA fragment from clone 10-4 wasinserted into pRc/CMV2 in the reverse (that is, non-translatable)orientation. The recombinant vector produced is designated pCMV2/RLMP.

An appropriate volume of pCMV10-4 (60 nM final concentration is optimal[3 μg]; for this experiment a range of 0-600 nM/well [0-30 μg/well]final concentration is preferred) was resuspended in Minimal Eagle Media(MEM) to 450 μl final volume and vortexed for 10 seconds. Superfect wasadded (7.5 μl/ml final solution), the solution was vortexed for 10seconds and then incubated at room temperature for 10 minutes. Followingthis incubation, MEM supplemented with 10% FBS (1 ml/well; 6 ml/plate)was added and mixed by pipetting.

The resulting solution was then promptly pipetted (1 ml/well) ontowashed ROB cultures. The cultures were incubated for 2 hours at 37° C.in a humidified atmosphere containing 5% CO₂. Afterward, the cells weregently washed once with sterile PBS and the appropriate normalincubation medium was added.

Results demonstrated significant bone nodule formation in all rat cellcultures which were induced with pCMV10-4. For example, PCMV10-4transfected cells produced 429 nodules/well. Positive control cultures,which were exposed to Trm, produced 460 nodules/well. In contrast,negative controls, which received no treatment, produced 1 nodule/well.Similarly, when cultures were transfected with pCMV10-4 (reverse), nonodules were observed.

For demonstrating de novo bone formation in vivo, marrow was aspiratedfrom the hindlimbs of 4-5 week old normal rats (mu/+; heterozygous forrecessive athymic condition). The aspirated marrow cells were washed inalpha MEM, centrifuged, and RBCs were lysed by resuspending the pelletin 0.83% NH₄Cl in 10 mM Tris (pH 7.4). The remaining marrow cells werewashed 3× with MEM and transfected for 2 hours with 9 μg of pCMV-LMP-1s(forward or reverse orientation) per 3×10⁶ cells. The transfected cellswere then washed 2× with MEM and resuspended at a concentration of 3×10⁷cells/ml.

The cell suspension (100 μl) was applied via sterile pipette to asterile 2×5 mm type I bovine collagen disc (Sulzer Orthopaedics, WheatRidge, CO). The discs were surgically implanted subcutaneously on theskull, chest, abdomen or dorsal spine of 4-5 week old athymic rats(mu/mu). The animals were scarified at 3-4 weeks, at which time thediscs or surgical areas were excised and fixed in 70% ethanol. The fixedspecimens were analyzed by radiography and undecalcified histologicexamination was performed on 5 μm thick sections stained with GoldnerTrichrome. Experiments were also performed using devitalized (guanidineextracted) demineralized bone matrix (Osteotech, Shrewsbury, N.J.) inplace of collagen discs.

Radiography revealed a high level of mineralized bone formation thatconformed to the form of the original collagen disc containing LMP-1stransfected marrow cells. No mineralized bone formation was observed inthe negative control (cells transfected with a reverse-oriented versionof the LMP-1s cDNA that did not code for a translated protein), andabsorption of the carrier appeared to be well underway.

Histology revealed new bone trabeculae lined with osteroblasts in theLMP-1s transfected implants. No bone was seen along With partialresorption of the carrier in the negative controls.

Radiography of a further experiment in which 18 sets (9 negative controlpCMV-LMP-REV & 9 experimental pCMV-LMP-1s) of implants were added tosites alternating between lumbar and thoracic spine in athymic ratsdemonstrated 0/9 negative control implants exhibiting bone formation(spine fusion) between vertebrae. All nine of the pCMV-LMP-1s treatedimplants exhibited solid bone fusions between vertebrae.

EXAMPLE 26 The Synthesis of pHIS-5′ ATG LMP-1s Expression Vector fromthe Sequences Demonstrated in Examples 2 and 3.

The 717 base-pair clone (Example 17) was digested with ClaI and EcoRV(New England Biologicals, city, Mass.). A small fragment (˜250base-pairs) was gel purified. Clone No. 7 (Example 18) was digested withClaI and XbaI. A 1400 base-pair fragment was gel purified from thatdigest. The isolated 250 base-pair and 1400 base-pair cDNA fragmentswere ligated by standard methods to form a fragment of ˜1650 bp. ThepHis-A vector (In Vitrogen) was digested with EcoRV and XbaI. Thelinearized vector was recovered and ligated to the chimeric 1650base-pair cDNA fragment. The ligated product was cloned and amplified bystandard methods, and the pHis-A-5′ ATG LMP-1s expression vector, alsodenominated as the vector pHis-A with insert HLMP-1s, was deposited atthe ATCC as previously described.

EXAMPLE 27 The Induction of Bone Nodule Formation and Mineralization InVitro With pHis-S ATG LMP-is Expression Vector

Rat calvarial cells were isolated and grown in secondary cultureaccording to Example 1. Cultures were either unstimulated or stimulatedwith glucocorticoid (GC) according to Example 1. The cultures weretransfected with 3 μg of recombinant pHis-A vector DNA/well as describedin Example 25. Mineralized nodules were visualized by Von Kossa stainingaccording to Example 3.

Human LMP-1s gene product overexpression alone (i.e., without GCstimulation) induced significant bone nodule formation (˜203nodules/well) in vitro. This is approximately 50% of the amount ofnodules produced by cells exposed to the GC positive control (˜412nodules/well). Similar results were obtained with cultures transfectedwith pHisA-LMP-Rat Expression vector (˜152 nodules/well) andpCMV2/LMP-Rat-Fwd (˜206 nodules/well). In contrast, the negative controlpCMV2/LMP-Rat-Rev yielded (˜2 nodules/well), while approximately 4nodules/well were seen in the untreated plates. These data demonstratethat the human LMP-1 cDNA was at least as osteoinductive as the ratLMP-1 cDNA in this model system. The effect in this experiment was lessthan that observed with GC stimulation; but in some the effect wascomparable.

EXAMPLE 28 LMP Induces Secretion of a Soluble Osteoinductive Factor

Overexpression of RLMP-1 or HLMP-1s in rat calvarial osteoblast culturesas described in Example 24 resulted in significantly greater noduleformation than was observed in the negative control. To study themechanism of action of LIM mineralization protein conditioned medium washarvested at different time points, concentrated to 10×, sterilefiltered, diluted to its original concentration in medium containingfresh serum, and applied for four days to untransfected cells.

Conditioned media harvested from cells transfected with RLMP-1 orHLMP-1s at day 4 was approximately as effective in inducing noduleformation as direct overexpression of RLMP-1 in transfected cells.Conditioned media from cells transfected with RLMP-1 or HLMP-1 in thereverse orientation had no apparent effect on nodule formation. Nor didconditioned media harvested from LMP-1 transfected cultures before day 4induce nodule formation. These data suggest that expression of LMP-1caused the synthesis and/or secretion of a soluble factor, which did notappear in culture medium in effectie amounts until 4 days posttransfection.

Since overexpression of rLMP-1 resulted in the secretion of anosteoinductive factor into the medium, Western blot analysis was used todetermine if LMP-1 protein was present in the medium. The presence ofrLMP-1 protein was assessed using antibody specific for LMP-1 (QDPDEE)and detected by conventional means. LMP-1 protein was found only in thecell layer of the culture and not detected in the medium.

Partial purification of the osteoinductive soluble factor wasaccomplished by standard 25% and 100% ammonium sulfate cuts followed byDE-52 anion exchange batch chromatography (100 mM or 500 mM NaCl). Allactivity was observed in the high ammonium sulfate, high NaCl fractions.Such localization is consistent with the possibility of a single factorbeing responsible for conditioning the medium.

All cited publications are hereby incorporated by reference in theirentirety.

While the foregoing specification teaches the principles of the presentinvention, with examples provided for the purpose of illustration, itwill be appreciated by one skilled in the art from reading thisdisclosure that various changes in form and detail can be made withoutdeparting from the true scope of the invention.

36 1 457 PRT Rattus norvegicus 1 Met Asp Ser Phe Lys Val Val Leu Glu GlyPro Ala Pro Trp Gly Phe 1 5 10 15 Arg Leu Gln Gly Gly Lys Asp Phe AsnVal Pro Leu Ser Ile Ser Arg 20 25 30 Leu Thr Pro Gly Gly Lys Ala Ala GlnAla Gly Val Ala Val Gly Asp 35 40 45 Trp Val Leu Ser Ile Asp Gly Glu AsnAla Gly Ser Leu Thr His Ile 50 55 60 Glu Ala Gln Asn Lys Ile Arg Ala CysGly Glu Arg Leu Ser Leu Gly 65 70 75 80 Leu Ser Arg Ala Gln Pro Ala GlnSer Lys Pro Gln Lys Ala Leu Thr 85 90 95 Pro Pro Ala Asp Pro Pro Arg TyrThr Phe Ala Pro Ser Ala Ser Leu 100 105 110 Asn Lys Thr Ala Arg Pro PheGly Ala Pro Pro Pro Thr Asp Ser Ala 115 120 125 Leu Ser Gln Asn Gly GlnLeu Leu Arg Gln Leu Val Pro Asp Ala Ser 130 135 140 Lys Gln Arg Leu MetGlu Asn Thr Glu Asp Trp Arg Pro Arg Pro Gly 145 150 155 160 Thr Gly GlnSer Arg Ser Phe Arg Ile Leu Ala His Leu Thr Gly Thr 165 170 175 Glu PheMet Gln Asp Pro Asp Glu Glu Phe Met Lys Lys Ser Ser Gln 180 185 190 ValPro Arg Thr Glu Ala Pro Ala Pro Ala Ser Thr Ile Pro Gln Glu 195 200 205Ser Trp Pro Gly Pro Thr Thr Pro Ser Pro Thr Ser Arg Pro Pro Trp 210 215220 Ala Val Asp Pro Ala Phe Ala Glu Arg Tyr Ala Pro Asp Lys Thr Ser 225230 235 240 Thr Val Leu Thr Arg His Ser Gln Pro Ala Thr Pro Thr Pro LeuGln 245 250 255 Asn Arg Thr Ser Ile Val Gln Ala Ala Ala Gly Gly Gly ThrGly Gly 260 265 270 Gly Ser Asn Asn Gly Lys Thr Pro Val Cys His Gln CysHis Lys Ile 275 280 285 Ile Arg Gly Arg Tyr Leu Val Ala Leu Gly His AlaTyr His Pro Glu 290 295 300 Glu Phe Val Cys Ser Gln Cys Gly Lys Val LeuGlu Glu Gly Gly Phe 305 310 315 320 Phe Glu Glu Lys Gly Ala Ile Phe CysPro Ser Cys Tyr Asp Val Arg 325 330 335 Tyr Ala Pro Ser Cys Ala Lys CysLys Lys Lys Ile Thr Gly Glu Ile 340 345 350 Met His Ala Leu Lys Met ThrTrp His Val Pro Cys Phe Thr Cys Ala 355 360 365 Ala Cys Lys Thr Pro IleArg Asn Arg Ala Phe Tyr Met Glu Glu Gly 370 375 380 Ala Pro Tyr Cys GluArg Asp Tyr Glu Lys Met Phe Gly Thr Lys Cys 385 390 395 400 Arg Gly CysAsp Phe Lys Ile Asp Ala Gly Asp Arg Phe Leu Glu Ala 405 410 415 Leu GlyPhe Ser Trp His Asp Thr Cys Phe Val Cys Ala Ile Cys Gln 420 425 430 IleAsn Leu Glu Gly Lys Thr Phe Tyr Ser Lys Lys Asp Lys Pro Leu 435 440 445Cys Lys Ser His Ala Phe Ser His Val 450 455 2 1696 DNA Rattus norvegicus2 gcacgaggat cccagcgcgg ctcctggagg ccgccaggca gccgcccagc cgggcattca 60ggagcaggta ccatggattc cttcaaggta gtgctggagg gacctgcccc ttggggcttc 120cgtctgcaag ggggcaagga cttcaacgtg cccctctcca tctctcggct cactcctgga 180ggcaaggccg cacaggccgg tgtggccgtg ggagactggg tactgagtat cgacggtgag 240aacgccggaa gcctcacaca cattgaagcc cagaacaaga tccgtgcctg tggggagcgc 300ctcagcctgg gtcttagcag agcccagcct gctcagagca aaccacagaa ggccctgacc 360cctcccgccg accccccgag gtacactttt gcaccaagcg cctccctcaa caagacggcc 420cggcccttcg gggcaccccc acctactgac agcgccctgt cgcagaatgg acagctgctc 480agacagctgg tccctgatgc cagcaagcag cggctgatgg agaatactga agactggcgc 540ccgcggccag ggacaggcca gtcccgttcc ttccgcatcc ttgctcacct cacgggcaca 600gagttcatgc aagacccgga tgaggaattc atgaagaagt caagccaggt gcccaggaca 660gaagccccag ccccagcctc aaccataccc caggaatcct ggcctggccc caccaccccc 720agccccacca gccgcccacc ctgggccgta gatcctgcat ttgctgagcg ctatgcccca 780gacaaaacca gcacagtgct gacccgacac agccagccag ccacacctac gcctctgcag 840aaccgcacct ccatagttca ggctgcagct ggagggggca caggaggagg cagcaacaat 900ggcaagacgc ctgtatgcca ccagtgccac aagatcatcc gcggccgata cctggtagca 960ctgggccacg cgtaccatcc tgaggaattt gtgtgcagcc agtgtgggaa ggtcctggaa 1020gagggtggct tcttcgagga gaagggagct atcttttgcc cctcctgcta tgatgtgcgc 1080tatgcaccca gctgtgccaa atgcaagaag aagatcactg gagagatcat gcatgcgctg 1140aagatgacct ggcatgttcc ctgcttcacc tgtgcagcct gcaaaacccc tatccgcaac 1200agggctttct acatggagga gggggctccc tactgcgagc gagattacga gaagatgttt 1260ggcacaaagt gtcgcggctg tgacttcaag atcgatgccg gggaccgttt cctggaagcc 1320ctgggtttca gctggcatga tacgtgtttt gtttgcgcaa tatgtcaaat caacttggaa 1380ggaaagacct tctactccaa gaaggacaag cccctgtgca agagccatgc cttttcccac 1440gtatgagcac ctcctcacac tactgccacc ctactctgcc agaagggtga taaaatgaga 1500gagctctctc tccctcgacc tttctgggtg gggctggcag ccattgtcct agccttggct 1560cctggccaga tcctggggct ccctcctcac agtccccttt cccacacttc ctccaccacc 1620accaccgtca ctcacaggtg ctagcctcct agccccagtt cactctggtg tcacaataaa 1680cctgtatgta gctgtg 1696 3 260 DNA Rattus norvegicus 3 ttctacatggaggagggggc tccctactgc gagcgagatt acgagaagat gtttggcaca 60 aagtgtcgcggctgtgactt caagatcgat gccggggacc gtttcctgga agccctgggt 120 ttcagctggcatgatacgtg ttttgtttgc gcaatatgtc aaatcaactt ggaaggaaag 180 accttctactccaagaagga caagcccctg tgcaagagcc atgccttttc ccacgtatga 240 gcacctcctcacactactgc 260 4 16 DNA Artificial Sequence Differential Display PCRPrimer 4 aagctttttt tttttg 16 5 13 DNA Artificial Sequence DifferentialDisplay PCR Primer 5 aagcttggct atg 13 6 223 DNA Rattus norvegicus 6atccttgctc acctcacggg caccgagttc atgcaagacc cggatgagga gcacctgaag 60aaatcaagcc aggtgcccag gacagaagcc ccagccccag cctcatctac accccaggag 120ccctggcctg gccctaccgc ccccagccct accagccgcc cgccctgggc tgtggaccct 180gcgtttgccg agcgctatgc cccagacaaa accagcacag tgc 223 7 717 DNA Homosapiens 7 atggattcct tcaaggtagt gctggagggg ccagcacctt ggggcttccggctgcaaggg 60 ggcaaggact tcaatgtgcc cctctccatt tcccggctca ctcctgggggcaaagcggcg 120 caggccggag tggccgtggg tgactgggtg ctgagcatcg atggcgagaatgcgggtagc 180 ctcacacaca tcgaagctca gaacaagatc cgggcctgcg gggagcgcctcagcctgggc 240 ctcagcaggg cccagccggt tcagagcaaa ccgcagaagg cctccgcccccgccgcggac 300 cctccgcggt acacctttgc acccagcgtc tccctcaaca agacggcccggccctttggg 360 gcgcccccgc ccgctgacag cgccccgcaa cagaatggac agccgctccgaccgctggtc 420 ccagatgcca gcaagcagcg gctgatggag aacacagagg actggcggccgcggccgggg 480 acaggccagt cgcgttcctt ccgcatcctt gcccacctca caggcaccgagttcatgcaa 540 gacccggatg aggagcacct gaagaaatca agccaggtgc ccaggacagaagccccagcc 600 ccagcctcat ctacacccca ggagccctgg cctggcccta ccgcccccagccctaccagc 660 cgcccgccct gggctgtgga ccctgcgttt gccgagcgct atgccccggacaaaacg 717 8 1488 DNA Homo sapiens 8 atcgatggcg agaatgcggg tagcctcacacacatcgaag ctcagaacaa gatccgggcc 60 tgcggggagc gcctcagcct gggcctcagcagggcccagc cggttcagag caaaccgcag 120 aaggcctccg cccccgccgc ggaccctccgcggtacacct ttgcacccag cgtctccctc 180 aacaagacgg cccggccctt tggggcgcccccgcccgctg acagcgcccc gcaacagaat 240 ggacagccgc tccgaccgct ggtcccagatgccagcaagc agcggctgat ggagaacaca 300 gaggactggc ggccgcggcc ggggacaggccagtcgcgtt ccttccgcat ccttgcccac 360 ctcacaggca ccgagttcat gcaagacccggatgaggagc acctgaagaa atcaagccag 420 gtgcccagga cagaagcccc agccccagcctcatctacac cccaggagcc ctggcctggc 480 cctaccgccc ccagccctac cagccgcccgccctgagctg tggaccctgc gtttgccgag 540 cgctatgccc cggacaaaac gagcacagtgctgacccggc acagccagcc ggccacgccc 600 acgccgctgc agagccgcac ctccattgtgcaggcagctg ccggaggggt gccaggaggg 660 ggcagcaaca acggcaagac tcccgtgtgtcaccagtgcc acaaggtcat ccggggccgc 720 tacctggtgg cgttgggcca cgcgtaccacccggaggagt ttgtgtgtag ccagtgtggg 780 aaggtcctgg aagagggtgg cttctttgaggagaagggcg ccatcttctg cccaccatgc 840 tatgacgtgc gctatgcacc cagctgtgccaagtgcaaga agaagattac aggcgagatc 900 atgcacgccc tgaagatgac ctggcacgtgcactgcttta cctgtgctgc ctgcaagacg 960 cccatccgga acagggcctt ctacatggaggagggcgtgc cctattgcga gcgagactat 1020 gagaagatgt ttggcacgaa atgccatggctgtgacttca agatcgacgc tggggaccgc 1080 ttcctggagg ccctgggctt cagctggcatgacacctgct tcgtctgtgc gatatgtcag 1140 atcaacctgg aaggaaagac cttctactccaagaaggaca ggcctctctg caagagccat 1200 gccttctctc atgtgtgagc cccttctgcccacagctgcc gcggtggccc ctagcctgag 1260 gggcctggag tcgtggccct gcatttctgggtagggctgg caatggttgc cttaaccctg 1320 gctcctggcc cgagcctggg ctcccgggcccctgcccacc caccttatcc tcccacccca 1380 ctccctccac caccacagca caccggtgctggccacacca gccccctttc acctccagtg 1440 ccacaataaa cctgtaccca gctgaattccaaaaaatcca aaaaaaaa 1488 9 1644 DNA Homo sapiens 9 atggattcct tcaaggtagtgctggagggg ccagcacctt ggggcttccg gctgcaaggg 60 ggcaaggact tcaatgtgcccctctccatt tcccggctca ctcctggggg caaagcggcg 120 caggccggag tggccgtgggtgactgggtg ctgagcatcg atggcgagaa tgcgggtagc 180 ctcacacaca tcgaagctcagaacaagatc cgggcctgcg gggagcgcct cagcctgggc 240 ctcagcaggg cccagccggttcagagcaaa ccgcagaagg cctccgcccc cgccgcggac 300 cctccgcggt acacctttgcacccagcgtc tccctcaaca agacggcccg gccctttggg 360 gcgcccccgc ccgctgacagcgccccgcaa cagaatggac agccgctccg accgctggtc 420 ccagatgcca gcaagcagcggctgatggag aacacagagg actggcggcc gcggccgggg 480 acaggccagt cgcgttccttccgcatcctt gcccacctca caggcaccga gttcatgcaa 540 gacccggatg aggagcacctgaagaaatca agccaggtgc ccaggacaga agccccagcc 600 ccagcctcat ctacaccccaggagccctgg cctggcccta ccgcccccag ccctaccagc 660 cgcccgccct gggctgtggaccctgcgttt gccgagcgct atgccccgga caaaacgagc 720 acagtgctga cccggcacagccagccggcc acgcccacgc cgctgcagag ccgcacctcc 780 attgtgcagg cagctgccggaggggtgcca ggagggggca gcaacaacgg caagactccc 840 gtgtgtcacc agtgccacaaggtcatccgg ggccgctacc tggtggcgtt gggccacgcg 900 taccacccgg aggagtttgtgtgtagccag tgtgggaagg tcctggaaga gggtggcttc 960 tttgaggaga agggcgccatcttctgccca ccatgctatg acgtgcgcta tgcacccagc 1020 tgtgccaagt gcaagaagaagattacaggc gagatcatgc acgccctgaa gatgacctgg 1080 cacgtgcact gctttacctgtgctgcctgc aagacgccca tccggaacag ggccttctac 1140 atggaggagg gcgtgccctattgcgagcga gactatgaga agatgtttgg cacgaaatgc 1200 catggctgtg acttcaagatcgacgctggg gaccgcttcc tggaggccct gggcttcagc 1260 tggcatgaca cctgcttcgtctgtgcgata tgtcagatca acctggaagg aaagaccttc 1320 tactccaaga aggacaggcctctctgcaag agccatgcct tctctcatgt gtgagcccct 1380 tctgcccaca gctgccgcggtggcccctag cctgaggggc ctggagtcgt ggccctgcat 1440 ttctgggtag ggctggcaatggttgcctta accctggctc ctggcccgag cctgggctcc 1500 cgggcccctg cccacccaccttatcctccc accccactcc ctccaccacc acagcacacc 1560 ggtgctggcc acaccagccccctttcacct ccagtgccac aataaacctg tacccagctg 1620 aattccaaaa aatccaaaaaaaaa 1644 10 457 PRT Homo sapiens 10 Met Asp Ser Phe Lys Val Val Leu GluGly Pro Ala Pro Trp Gly Phe 1 5 10 15 Arg Leu Gln Gly Gly Lys Asp PheAsn Val Pro Leu Ser Ile Ser Arg 20 25 30 Leu Thr Pro Gly Gly Lys Ala AlaGln Ala Gly Val Ala Val Gly Asp 35 40 45 Trp Val Leu Ser Ile Asp Gly GluAsn Ala Gly Ser Leu Thr His Ile 50 55 60 Glu Ala Gln Asn Lys Ile Arg AlaCys Gly Glu Arg Leu Ser Leu Gly 65 70 75 80 Leu Ser Arg Ala Gln Pro ValGln Ser Lys Pro Gln Lys Ala Ser Ala 85 90 95 Pro Ala Ala Asp Pro Pro ArgTyr Thr Phe Ala Pro Ser Val Ser Leu 100 105 110 Asn Lys Thr Ala Arg ProPhe Gly Ala Pro Pro Pro Ala Asp Ser Ala 115 120 125 Pro Gln Gln Asn GlyGln Pro Leu Arg Pro Leu Val Pro Asp Ala Ser 130 135 140 Lys Gln Arg LeuMet Glu Asn Thr Glu Asp Trp Arg Pro Arg Pro Gly 145 150 155 160 Thr GlyGln Ser Arg Ser Phe Arg Ile Leu Ala His Leu Thr Gly Thr 165 170 175 GluPhe Met Gln Asp Pro Asp Glu Glu His Leu Lys Lys Ser Ser Gln 180 185 190Val Pro Arg Thr Glu Ala Pro Ala Pro Ala Ser Ser Thr Pro Gln Glu 195 200205 Pro Trp Pro Gly Pro Thr Ala Pro Ser Pro Thr Ser Arg Pro Pro Trp 210215 220 Ala Val Asp Pro Ala Phe Ala Glu Arg Tyr Ala Pro Asp Lys Thr Ser225 230 235 240 Thr Val Leu Thr Arg His Ser Gln Pro Ala Thr Pro Thr ProLeu Gln 245 250 255 Ser Arg Thr Ser Ile Val Gln Ala Ala Ala Gly Gly ValPro Gly Gly 260 265 270 Gly Ser Asn Asn Gly Lys Thr Pro Val Cys His GlnCys His Lys Val 275 280 285 Ile Arg Gly Arg Tyr Leu Val Ala Leu Gly HisAla Tyr His Pro Glu 290 295 300 Glu Phe Val Cys Ser Gln Cys Gly Lys ValLeu Glu Glu Gly Gly Phe 305 310 315 320 Phe Glu Glu Lys Gly Ala Ile PheCys Pro Pro Cys Tyr Asp Val Arg 325 330 335 Tyr Ala Pro Ser Cys Ala LysCys Lys Lys Lys Ile Thr Gly Glu Ile 340 345 350 Met His Ala Leu Lys MetThr Trp His Val His Cys Phe Thr Cys Ala 355 360 365 Ala Cys Lys Thr ProIle Arg Asn Arg Ala Phe Tyr Met Glu Glu Gly 370 375 380 Val Pro Tyr CysGlu Arg Asp Tyr Glu Lys Met Phe Gly Thr Lys Cys 385 390 395 400 His GlyCys Asp Phe Lys Ile Asp Ala Gly Asp Arg Phe Leu Glu Ala 405 410 415 LeuGly Phe Ser Trp His Asp Thr Cys Phe Val Cys Ala Ile Cys Gln 420 425 430Ile Asn Leu Glu Gly Lys Thr Phe Tyr Ser Lys Lys Asp Arg Pro Leu 435 440445 Cys Lys Ser His Ala Phe Ser His Val 450 455 11 22 DNA ArtificialSequence Sequencing Primer 11 gccagggttt tcccagtcac ga 22 12 22 DNAArtificial Sequence Sequencing Primer 12 gccagggttt tcccagtcac ga 22 1322 DNA Homo sapiens 13 tcttagcaga gcccagcctg ct 22 14 22 DNA Homosapiens 14 gcatgaactc tgtgcccgtg ag 22 15 20 DNA Rattus norvegicus 15atccttgctc acctcacggg 20 16 22 DNA Rattus norvegicus 16 gcactgtgctggttttgtct gg 22 17 23 DNA Homo sapiens 17 catggattcc ttcaaggtag tgc 2318 20 DNA Homo sapiens 18 gttttgtctg gggcagagcg 20 19 44 DNA ArtificialSequence Sequencing Primer 19 ctaatacgac tcactatagg gctcgagcggccgcccgggc aggt 44 20 27 DNA Artificial Sequence Sequencing Primer 20ccatcctaat acgactcact atagggc 27 21 765 DNA Homo sapiens 21 ccgttgtttgtaaaacgacg cagagcagcg ccctggccgg gccaagcagg agccggcatc 60 atggattccttcaaggtagt gctggagggg ccagcacctt ggggcttccg gctgcaaggg 120 ggcaaggacttcaatgtgcc ctcctccatt tcccggctca cctctggggg caaggccgtg 180 caggccggagtggccgtaag tgactgggtg ctgagcatcg atggcgagaa tgcgggtagc 240 ctcacacacatcgaagctca gaacaagatc cgggcctgcg gggagcgcct cagcctgggc 300 ctcaacagggcccagccggt tcagaacaaa ccgcaaaagg cctccgcccc cgccgcggac 360 cctccgcggtacacctttgc accaagcgtc tccctcaaca agacggcccg gcccttgggg 420 gcgcccccgcccgctgacag cgccccgcag cagaatggac agccgctccg accgctggtc 480 ccagatgccagcaagcagcg gctgatggag aacacagagg actggcggcc gcggccgggg 540 acaggccagtgccgttcctt tcgcatcctt gctcacctta caggcaccga gttcatgcaa 600 gacccggatgaggagcacct gaagaaatca agccaggtgc ccaggacaga agccccagcc 660 ccagcctcatctacacccca ggagccctgg cctggcccta ccgcccccag ccctaccagc 720 cgcccgccctgggctgtgga ccctgcgttt gccgagcgct atgcc 765 22 1689 DNA Homo sapiens 22cgacgcagag cagcgccctg gccgggccaa gcaggagccg gcatcatgga ttccttcaag 60gtagtgctgg aggggccagc accttggggc ttccggctgc aagggggcaa ggacttcaat 120gtgcccctct ccatttcccg gctcactcct gggggcaaag cggcgcaggc cggagtggcc 180gtgggtgact gggtgctgag catcgatggc gagaatgcgg gtagcctcac acacatcgaa 240gctcagaaca agatccgggc ctgcggggag cgcctcagcc tgggcctcag cagggcccag 300ccggttcaga gcaaaccgca gaaggcctcc gcccccgccg cggaccctcc gcggtacacc 360tttgcaccca gcgtctccct caacaagacg gcccggccct ttggggcgcc cccgcccgct 420gacagcgccc cgcaacagaa tggacagccg ctccgaccgc tggtcccaga tgccagcaag 480cagcggctga tggagaacac agaggactgg cggccgcggc cggggacagg ccagtcgcgt 540tccttccgca tccttgccca cctcacaggc accgagttca tgcaagaccc ggatgaggag 600cacctgaaga aatcaagcca ggtgcccagg acagaagccc cagccccagc ctcatctaca 660ccccaggagc cctggcctgg ccctaccgcc cccagcccta ccagccgccc gccctgggct 720gtggaccctg cgtttgccga gcgctatgcc ccggacaaaa cgagcacagt gctgacccgg 780cacagccagc cggccacgcc cacgccgctg cagagccgca cctccattgt gcaggcagct 840gccggagggg tgccaggagg gggcagcaac aacggcaaga ctcccgtgtg tcaccagtgc 900cacaaggtca tccggggccg ctacctggtg gcgttgggcc acgcgtacca cccggaggag 960tttgtgtgta gccagtgtgg gaaggtcctg gaagagggtg gcttctttga ggagaagggc 1020gccatcttct gcccaccatg ctatgacgtg cgctatgcac ccagctgtgc caagtgcaag 1080aagaagatta caggcgagat catgcacgcc ctgaagatga cctggcacgt gcactgcttt 1140acctgtgctg cctgcaagac gcccatccgg aacagggcct tctacatgga ggagggcgtg 1200ccctattgcg agcgagacta tgagaagatg tttggcacga aatgccatgg ctgtgacttc 1260aagatcgacg ctggggaccg cttcctggag gccctgggct tcagctggca tgacacctgc 1320ttcgtctgtg cgatatgtca gatcaacctg gaaggaaaga ccttctactc caagaaggac 1380aggcctctct gcaagagcca tgccttctct catgtgtgag ccccttctgc ccacagctgc 1440cgcggtggcc cctagcctga ggggcctgga gtcgtggccc tgcatttctg ggtagggctg 1500gcaatggttg ccttaaccct ggctcctggc ccgagcctgg gctcccgggc ccctgcccac 1560ccaccttatc ctcccacccc actccctcca ccaccacagc acaccggtgc tggccacacc 1620agcccccttt cacctccagt gccacaataa acctgtaccc agctgaattc caaaaaatcc 1680aaaaaaaaa 1689 23 22 DNA Homo sapiens 23 gcactgtgct cgttttgtcc gg 22 2421 DNA Homo sapiens 24 tccttgctca cctcacgggc a 21 25 30 DNA Homo sapiens25 tcctcatccg ggtcttgcat gaactcggtg 30 26 28 DNA Homo sapiens Sequencingprimer 26 gcccccgccc gctgacagcg ccccgcaa 28 27 24 DNA Homo sapiens 27tccttgctca cctcacgggc accg 24 28 22 DNA Artificial Sequence SequencingPrimer 28 gtaatacgac tcactatagg gc 22 29 23 DNA Rattus norvegicus 29gcggctgatg gagaatactg aag 23 30 23 DNA Rattus norvegicus 30 atcttgtggcactggtggca tac 23 31 22 DNA Rattus norvegicus 31 tgtgtcgggt cagcactgtgct 22 32 1620 DNA Homo sapiens 32 atggattcct tcaaggtagt gctggaggggccagcacctt ggggcttccg gctgcaaggg 60 ggcaaggact tcaatgtgcc cctctccatttcccggctca ctcctggggg caaagcggcg 120 caggccggag tggccgtggg tgactgggtgctgagcatcg atggcgagaa tgcgggtagc 180 ctcacacaca tcgaagctca gaacaagatccgggcctgcg gggagcgcct cagcctgggc 240 ctcagcaggg cccagccggt tcagagcaaaccgcagaagg cctccgcccc cgccgcggac 300 cctccgcggt acacctttgc acccagcgtctccctcaaca agacggcccg gccctttggg 360 gcgcccccgc ccgctgacag cgccccgcaacagaatggac agccgctccg accgctggtc 420 ccagatgcca gcaagcagcg gctgatggagaacacagagg actggcggcc gcggccgggg 480 acaggccagt cgcgttcctt ccgcatccttgcccacctca caggcaccga gttcatgcaa 540 gacccggatg aggagcacct gaagaaatcaagccaggtgc ccaggacaga agccccagcc 600 ccagcctcat ctacacccca ggagccctggcctggcccta ccgcccccag ccctaccagc 660 cgcccgccct gagctgtgga ccctgcgtttgccgagcgct atgccccgga caaaacgagc 720 acagtgctga cccggcacag ccagccggccacgcccacgc cgctgcagag ccgcacctcc 780 attgtgcagg cagctgccgg aggggtgccaggagggggca gcaacaacgg caagactccc 840 gtgtgtcacc agtgccacaa ggtcatccggggccgctacc tggtggcgtt gggccacgcg 900 taccacccgg aggagtttgt gtgtagccagtgtgggaagg tcctggaaga gggtggcttc 960 tttgaggaga agggcgccat cttctgcccaccatgctatg acgtgcgcta tgcacccagc 1020 tgtgccaagt gcaagaagaa gattacaggcgagatcatgc acgccctgaa gatgacctgg 1080 cacgtgcact gctttacctg tgctgcctgcaagacgccca tccggaacag ggccttctac 1140 atggaggagg gcgtgcccta ttgcgagcgagactatgaga agatgtttgg cacgaaatgc 1200 catggctgtg acttcaagat cgacgctggggaccgcttcc tggaggccct gggcttcagc 1260 tggcatgaca cctgcttcgt ctgtgcgatatgtcagatca acctggaagg aaagaccttc 1320 tactccaaga aggacaggcc tctctgcaagagccatgcct tctctcatgt gtgagcccct 1380 tctgcccaca gctgccgcgg tggcccctagcctgaggggc ctggagtcgt ggccctgcat 1440 ttctgggtag ggctggcaat ggttgccttaaccctggctc ctggcccgag cctgggctcc 1500 cgggcccctg cccacccacc ttatcctcccaccccactcc ctccaccacc acagcacacc 1560 ggtgctggcc acaccagccc cctttcacctccagtgccac aataaacctg tacccagctg 1620 33 1665 DNA Homo sapiens 33cgacgcagag cagcgccctg gccgggccaa gcaggagccg gcatcatgga ttccttcaag 60gtagtgctgg aggggccagc accttggggc ttccggctgc aagggggcaa ggacttcaat 120gtgcccctct ccatttcccg gctcactcct gggggcaaag cggcgcaggc cggagtggcc 180gtgggtgact gggtgctgag catcgatggc gagaatgcgg gtagcctcac acacatcgaa 240gctcagaaca agatccgggc ctgcggggag cgcctcagcc tgggcctcag cagggcccag 300ccggttcaga gcaaaccgca gaaggcctcc gcccccgccg cggaccctcc gcggtacacc 360tttgcaccca gcgtctccct caacaagacg gcccggccct ttggggcgcc cccgcccgct 420gacagcgccc cgcaacagaa tggacagccg ctccgaccgc tggtcccaga tgccagcaag 480cagcggctga tggagaacac agaggactgg cggccgcggc cggggacagg ccagtcgcgt 540tccttccgca tccttgccca cctcacaggc accgagttca tgcaagaccc ggatgaggag 600cacctgaaga aatcaagcca ggtgcccagg acagaagccc cagccccagc ctcatctaca 660ccccaggagc cctggcctgg ccctaccgcc cccagcccta ccagccgccc gccctgagct 720gtggaccctg cgtttgccga gcgctatgcc ccggacaaaa cgagcacagt gctgacccgg 780cacagccagc cggccacgcc cacgccgctg cagagccgca cctccattgt gcaggcagct 840gccggagggg tgccaggagg gggcagcaac aacggcaaga ctcccgtgtg tcaccagtgc 900cacaaggtca tccggggccg ctacctggtg gcgttgggcc acgcgtacca cccggaggag 960tttgtgtgta gccagtgtgg gaaggtcctg gaagagggtg gcttctttga ggagaagggc 1020gccatcttct gcccaccatg ctatgacgtg cgctatgcac ccagctgtgc caagtgcaag 1080aagaagatta caggcgagat catgcacgcc ctgaagatga cctggcacgt gcactgcttt 1140acctgtgctg cctgcaagac gcccatccgg aacagggcct tctacatgga ggagggcgtg 1200ccctattgcg agcgagacta tgagaagatg tttggcacga aatgccatgg ctgtgacttc 1260aagatcgacg ctggggaccg cttcctggag gccctgggct tcagctggca tgacacctgc 1320ttcgtctgtg cgatatgtca gatcaacctg gaaggaaaga ccttctactc caagaaggac 1380aggcctctct gcaagagcca tgccttctct catgtgtgag ccccttctgc ccacagctgc 1440cgcggtggcc cctagcctga ggggcctgga gtcgtggccc tgcatttctg ggtagggctg 1500gcaatggttg ccttaaccct ggctcctggc ccgagcctgg gctcccgggc ccctgcccac 1560ccaccttatc ctcccacccc actccctcca ccaccacagc acaccggtgc tggccacacc 1620agcccccttt cacctccagt gccacaataa acctgtaccc agctg 1665 34 223 PRT Homosapiens 34 Met Asp Ser Phe Lys Val Val Leu Glu Gly Pro Ala Pro Trp GlyPhe 1 5 10 15 Arg Leu Gln Gly Gly Lys Asp Phe Asn Val Pro Leu Ser IleSer Arg 20 25 30 Leu Thr Pro Gly Gly Lys Ala Ala Gln Ala Gly Val Ala ValGly Asp 35 40 45 Trp Val Leu Ser Ile Asp Gly Glu Asn Ala Gly Ser Leu ThrHis Ile 50 55 60 Glu Ala Gln Asn Lys Ile Arg Ala Cys Gly Glu Arg Leu SerLeu Gly 65 70 75 80 Leu Ser Arg Ala Gln Pro Val Gln Ser Lys Pro Gln LysAla Ser Ala 85 90 95 Pro Ala Ala Asp Pro Pro Arg Tyr Thr Phe Ala Pro SerVal Ser Leu 100 105 110 Asn Lys Thr Ala Arg Pro Phe Gly Ala Pro Pro ProAla Asp Ser Ala 115 120 125 Pro Gln Gln Asn Gly Gln Pro Leu Arg Pro LeuVal Pro Asp Ala Ser 130 135 140 Lys Gln Arg Leu Met Glu Asn Thr Glu AspTrp Arg Pro Arg Pro Gly 145 150 155 160 Thr Gly Gln Ser Arg Ser Phe ArgIle Leu Ala His Leu Thr Gly Thr 165 170 175 Glu Phe Met Gln Asp Pro AspGlu Glu His Leu Lys Lys Ser Ser Gln 180 185 190 Val Pro Arg Thr Glu AlaPro Ala Pro Ala Ser Ser Thr Pro Gln Glu 195 200 205 Pro Trp Pro Gly ProThr Ala Pro Ser Pro Thr Ser Arg Pro Pro 210 215 220 35 25 DNA Rattusnorvegicus 35 gcactacctt gaaggaatcc atggt 25 36 6 PRT Rattus norvegicus36 Gln Asp Pro Asp Glu Glu 1 5

We claim:
 1. A method of fusing a spine, comprising: (a) transfectingosteogenic precursor cells with an isolated nucleic acid moleculecomprising a nucleotide sequence encoding LIM mineralization protein,wherein said nucleic acid molecule is SEQ ID NO: 22 or SEQ ID NO: 33;(b) admixing the transfected osteogenic precursor cells with a matrix;and (c) contacting the matrix with the spine wherein the expression ofthe nucleotide sequence encoding LIM mineralization protein causesmineralized bone formation in the matrix, whereby the spine is fused. 2.The method of claim 1, wherein the osteogenic precursor cells aretransfected ex vivo.
 3. The method of claim 1, wherein the LIMmineralization protein is HLMP-1 or HLMP-1s.
 4. The method of claim 1,wherein the osteogenic precursor cells are mammalian cells.
 5. A methodof fusing a spine, comprising: (a) transfecting osteogenic precursorcells with an isolated nucleic acid molecule comprising a nucleotidesequence encoding a LIM mineralization protein, wherein the nucleic acidmolecule hybridizes under standard conditions to a nucleic acid moleculecomplementary to the full length of SEQ ID NO: 25 or the moleculehybridizes under highly stringent conditions to a nucleic acid moleculecomplementary to the full length of SEQ ID NO: 26, and wherein saidmolecule includes the sequences of nucleotides shown in SEQ ID Nos: 25and 26; (b) admixing the transfected osteogenic precursor cells with amatrix; and (c) contacting the matrix with the spine wherein theexpression of the nucleotide sequence encoding LIM mineralizationprotein causes mineralized bone formation in the matrix, whereby thespine is fused.
 6. The method of claim 5, wherein the osteogenicprecursor cells are transfected ex vivo.
 7. The method of claim 5,wherein the isolated nucleic acid molecule is HLMP-1s which comprisesSEQ ID NO:
 33. 8. The method of claim 5, wherein the isolated nucleicacid molecule is HLMP-1 which comprises SEQ ID NO:
 22. 9. A method offusing a spine, comprising: (a) transfecting osteogenic precursor cellswith an isolated nucleic acid molecule comprising a nucleotide sequenceencoding a LIM mineralization protein, wherein the nucleic acid moleculehybridizes under standard conditions to a nucleic acid moleculecomplementary to the full length of SEQ ID NO: 25, and wherein saidmolecule includes the sequence of nucleotides shown in SEQ ID No: 25.(b) admixing the transfected osteogenic precursor cells with a matrix;and (c) contacting the matrix with the spine wherein the expression ofthe nucleotide sequence encoding LIM mineralization protein causesmineralized bone formation in the matrix, whereby the spine is fused.10. The method of claim 9, wherein the osteogenic precursor cells aretransfected ex vivo.
 11. The method of claim 9, wherein the osteogenicprecursor cells are mammalian cells.
 12. A method of fusing a spine,comprising: (a) transfecting osteogenic precursor cells with an isolatednudleic acid molecule comprising a nucleotide sequence encoding a LIMmineralization protein, wherein the nucleic acid molecule hybridizesunder standard conditions to a nucleic acid molecule complementary tothe full length of SEQ ID NO: 26, and wherein said molecule includes thesequence of nucleotides shown in SEQ ID No: 26 (b) admixing thetransfected osteogenic precursor cells with a matrix; and (c) contactingthe matrix with the spine wherein the expression of the nucleotidesequence encoding LIM mineralization protein causes mineralized boneformation in the matrix, whereby the spine is fused.
 13. The method ofclaim 12, wherein the osteogenic precursor cells are transfected exvivo.
 14. The method of claim 12, wherein the osteogenic precursor cellsare mammalian cells.